System, method, and product for scanning of biological materials

ABSTRACT

An embodiment of a scanning system is described including optical elements that direct an excitation beam at a probe array, detectors that receive reflected intensity data responsive to the excitation beam, where the reflected intensity data is responsive to a focusing distance between an optical element and the probe array, a transport frame that adjusts the focusing distance in a direction with respect to the probe array, an auto-focuser that determines a best plane of focus based upon characteristics of the reflected intensity data of at least two focusing distances where the detectors further receive pixel intensity values based upon detected emissions from a plurality of probe features disposed on the probe array at the best plane of focus, and an image generator that associates each of the pixel intensity values with at least one image pixel position of a probe array based upon one or more position correction values.

RELATED APPLICATIONS

The present application claims priority from U.S. Provisional PatentApplication Ser. No. 60/364,731, entitled “SYSTEM, METHOD, AND PRODUCTFOR SCANNING OF BIOLOGICAL MATERIALS,” filed Mar. 15, 2002; U.S.Provisional Patent Application Ser. No. 60/396,457, titled“HIGH-THROUGHPUT MICROARRAY SCANNING SYSTEM AND METHOD”, filed Jul. 17,2002; and U.S. Provisional Patent Application Ser. No. 60/435,178,titled “System, Method and Product for Scanning of BiologicalMaterials”, flied Dec. 19, 2002, each of which is hereby incorporated byreference herein in it's entirety for all purposes.

BACKGROUND

1. Field of the Invention

The present invention relates to system and methods for biologicalmaterial. In particular, the invention relates to improved opticalreaders or scanners for detecting emissions from biological probe arrayshaving small features that may be arranged in high densities on thearrays.

2. Related Art

Synthesized nucleic acid probe arrays, such as Affymetrix GeneChip®probe arrays, and spotted probe arrays, have been used to generateunprecedented amounts of information about biological systems. Forexample, the GeneChip® Human Genome U133 Set (HG-U133A and HG-U133B)available from Affymetrix, Inc. of Santa Clara, Calif., is comprised oftwo microarrays containing over 1,000,000 unique oligonucleotidefeatures covering more than 39,000 transcript variants that representmore than 33,000 human genes. Analysis of expression data from suchmicroarrays may lead to the development of new drugs and new diagnostictools.

SUMMARY OF THE INVENTION

Systems, methods, and products to address these and other needs aredescribed herein with respect to illustrative, non-limiting,implementations. Various alternatives, modifications and equivalents arepossible. For example, certain systems, methods, and computer softwareproducts are described herein using exemplary implementations foranalyzing data from arrays of biological materials produced by theAffymetrix® 417™ or 427™ Arrayer. Other illustrative implementations arereferred to in relation to data from Affymetrix® GeneChip® probe arrays.However, these systems, methods, and products may be applied withrespect to many other types of probe arrays and, more generally, withrespect to numerous parallel biological assays produced in accordancewith other conventional technologies and/or produced in accordance withtechniques that may be developed in the future. For example, thesystems, methods, and products described herein may be applied toparallel assays of nucleic acids, PCR products generated from cDNAclones, proteins, antibodies, or many other biological materials. Thesematerials may be disposed on slides (as typically used for spottedarrays), on substrates employed for GeneChip® arrays, or on beads,optical fibers, or other substrates or media, which may includepolymeric coatings or other layers on top of slides or other substrates.Moreover, the probes need not be immobilized in or on a substrate, and,if immobilized, need not be disposed in regular patterns or arrays. Forconvenience, the term “probe array” will generally be used broadlyhereafter to refer to all of these types of arrays and parallelbiological assays.

In accordance with a particular embodiment, a scanning system isdescribed including optical elements that direct an excitation beam at aprobe array, detectors that receive reflected intensity data responsiveto the excitation beam, where the reflected intensity data is responsiveto a focusing distance between an optical element and the probe array, atransport frame that adjusts the focusing distance in a direction withrespect to the probe array, an auto-focuser that determines a best planeof focus based upon characteristics of the reflected intensity data ofat least two focusing distances where the detectors further receivepixel intensity values based upon detected emissions from a plurality ofprobe features disposed on the probe array at the best plane of focus,and an image generator that associates each of the pixel intensityvalues with at least one image pixel position of a probe array basedupon one or more position correction values.

In accordance with another embodiment a method is described thatincludes the acts of receiving pixel intensity values based upondetected emissions from probe features disposed on a probe array, andassociating each of the pixel intensity values with image pixelpositions of a probe array image based upon position correction values.

In some implementations, the probe array image has a position error ofplus or minus one pixel or less, and the emissions are responsive to anexcitation beam and arise from excitation by the excitation beam offluorescent molecules. The excitation beam includes a laser beamgenerated by a solid state, diode pumped, frequency doubled Nd: YAGlaser. The act of receiving pixel intensity values is accomplished in asingle pass of the excitation beam over a number of contiguous probefeatures of the plurality of probe features.

The contiguous features may include probe features that are immediatelyadjacent to each other or are separated by non-feature areas of theprobe array. A scanning line may comprise a number of contiguousfeatures. Additionally, each of the pixel intensity values may beassociated with a pixel having a dimension in an x-axis with respect tothe probe array of approximately 2.5 μm, or in a range between about 1.5μM and about 3.0 μm. Also, each of the probe features has a dimension inan x-axis with respect to the probe array of approximately 18 μm, or ina range of between about 14 μm and about 22 μm. Similarly, each of theprobe features may have a dimension in an x-axis with respect to theprobe array of approximately 10 μm, or in a range of about 6 μm andabout 14 μm. Each probe feature may be associated with one or more ofthe pixel intensity values.

Also in some implementations, the biological probe array includes asynthesized array or a spotted array, and the position correction valueseach include a horizontal linearity correction value, a verticallinearity correction value, or both. The act of associating each of thepixel intensity values with image pixel positions is accomplished byadjusting a received pixel position associated with each of the pixelintensity values by a number of pixels determined by the one or moreposition correction values, where the number of pixels includes afractional value and the one or more position correction values may bebased upon one or more reference positions provided by one or morecalibration features. The one or more calibration features comprises anarray of features oriented in a horizontal pattern, vertical pattern, orboth.

In some embodiments the method further comprises the acts of translatingthe probe array a distance along a y-axis with respect to the probearray, and repeating the steps of receiving, associating, andtranslating until the probe array image includes pixel intensity valuescorresponding to each of the plurality of probes disposed on the probearray. The distance may based, at least in part, on a size of a pixel inthe y-axis, and include a distance of approximately 2.5 μm, or include adistance in a range between about 1.5 μm and about 3.0 μm.

In accordance with other embodiments a scanning system is described,including one or more detectors that receive a plurality of pixelintensity values based upon detected emissions from probe featuresdisposed on a probe array, and a corrected image generator thatassociates each of the pixel intensity values with one or more imagepixel positions of a probe array image based upon one or more positioncorrection values.

In some implementations, the scanning further includes a transport framethat translates the probe array a distance along a y-axis with respectto the probe array, and a comparator that is determine a completed probearray image based, at least in part, upon one or more received pixelintensity values corresponding to each of the plurality of probesdisposed on the probe array. The transport frame further provides asolid hinge flexure that may provide movement in the Z-axis, and has nofriction or stiction.

In accordance with yet another embodiment, a method is describedincluding the acts of directing an excitation beam at a probe array,receiving reflected intensity data responsive to the excitation beam,where the intensity data is responsive to a focusing distance between anoptical element and the probe array, adjusting the focusing distance ina direction with respect to the probe array, repeating the steps ofreceiving and adjusting for a number of iterations, and determining abest plane of focus based upon one or more characteristics of thereflected intensity data at the adjusted focusing distances.

In some implementations the direction is away from or toward the probearray, the number of iterations may be predetermined. The predeterminednumber of iterations is based on an anticipated error associated withthe reflected intensity data where the anticipated error may beinversely related to the predetermined number of iterations. Also, thereflected intensity data may be responsive to reflection of theexcitation beam from one or more focus features, and may correspond toone or more reflection spots. The best plane of focus may be based uponassociating the one or more spots with one or more characteristics of abeam waist. Additionally, the one or more focus features may bepositioned outside an active area and the one or more probe features arepositioned inside the active area.

Also, in some implementations the one or more focus features may includea chrome border, and the one or more characteristics may include a slopevalue. The best plane of focus may be based upon a maximum value of theslope value.

In accordance with another embodiment, a scanning system is described,including one or more optical elements that direct an excitation beam ata probe array, one or more detectors that receive reflected intensitydata responsive to the excitation beam where the intensity data may bedetermined by a focusing distance between an optical element and theprobe array, and an auto-focuser constructed and arranged to determine abest plane of focus based upon one or more characteristics of thereflected intensity data as received at two or more focusing distances.

In accordance with a further embodiment, a method is described includingthe acts of storing one or more probe arrays in a magazine where each ofthe plurality of probe arrays is housed in a probe array cartridge,reversibly transporting a first probe array cartridge between themagazine and a scanning system, and advancing the magazine by one ormore positions to a second probe array cartridge.

In some implementations the act of storing includes providing atemperature and humidity controlled environment that includesmaintaining the probe arrays at a temperature for maintaining biologicalintegrity. The temperature for maintaining biological integrity mayincludes a range between about 2° C. and 15° C. Also each of the one ormore probe arrays may be arranged in a vertical orientation, and themagazine holds a at least 48 or 100 probe arrays. The magazine issubstantially circular.

Additionally, in some implementations condensation on at least one ofthe probe array cartridges may be reduced by warming. The act oftransporting may also include one or more elements that reversiblyengage and disengage one or more fiducial features of the probe arraycartridge.

In accordance with an additional embodiment, a scanning system isdescribed, including a cooled storage chamber that stores one or moreprobe arrays in a magazine where each of the probe arrays is housed in aprobe array cartridge, a cartridge transport assembly that reversiblytransports a first probe array cartridge between the magazine and ascanning system, and a linear motor that advances the magazine by one ormore positions to a second probe array cartridge.

In accordance with another embodiment, a scanning system is describedincluding a service application that performs one or more calibrationmethods that also includes an interface to elements of the scanningsystem.

In some implementations, the one or more calibration methods includepitch calibration, roll calibration, and arc radius calibration, and theelements includes hardware elements. The service application is furtherruns one or more diagnostic tests, and uploads and/or downloads one ormore software or firmware applications.

The above embodiments and implementations are not necessarily inclusiveor exclusive of each other and may be combined in any manner that isnon-conflicting and otherwise possible, whether they be presented inassociation with a same, or a different, embodiment or implementation.The description of one embodiment or implementation is not intended tobe limiting with respect to other embodiments and/or implementations.Also, any one or more function, step, operation, or technique describedelsewhere in this specification may, in alternative implementations, becombined with any one or more function, step, operation, or techniquedescribed in the summary. Thus, the above embodiment and implementationsare illustrative rather than limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and further features will be more clearly appreciated from thefollowing detailed description when taken in conjunction with theaccompanying drawings. In the drawings, like reference numerals indicatelike structures or method steps and the leftmost digit of a referencenumeral indicates the number of the figure in which the referencedelement first appears (for example, the element 160 appears first inFIG. 1). In functional block diagrams, rectangles generally indicatefunctional elements and parallelograms generally indicate data. Inmethod flow charts, rectangles generally indicate method steps anddiamond shapes generally indicate decision elements. All of theseconventions, however, are intended to be typical or illustrative, ratherthan limiting.

FIG. 1 is a simplified graphical representation of an arrangement ofscanner optics and detectors suitable for providing excitation andemission signals;

FIG. 2A is a perspective view of a simplified exemplary configuration ofa scanning arm portion of the scanner optics and detectors of FIG. 1;

FIG. 2B is a top planar view of the scanning arm of FIG. 2A as it scansbiological features on one embodiment of a probe array being moved by atranslation stage under the arm's arcuate path;

FIG. 3 is a functional block diagram of one embodiment of a computernetwork system including user computers coupled to a server suitable forexecution of information management, and array-image-acquisition andanalysis, software applications in accordance with one embodiment of thepresent invention;

FIG. 4 is a functional block diagram of the server of FIG. 3 includingillustrative embodiments of the software application, auto-loader,scanner, and connections to user computers;

FIG. 5 is a functional block diagram of one embodiment of thescanner-computer system of FIG. 4 including a barcode reader, acartridge transport frame, a scanner computer, and scanner controloperations;

FIG. 6 is a functional block diagram of one embodiment of the scannercomputer of FIG. 5 including scanner firmware, auto-focus/auto-zerosoftware, calibration software, and service application software;

FIG. 7A is a graphical representation of one embodiment of the probearray of FIG. 2 housed in a cartridge, and illustrative examples of axesof movement;

FIG. 7B is a graphical representation of one embodiment of a probe arraycartridge such as in FIG. 7A including examples of an arcuate pathcrossing a chrome border for auto-focus methods;

FIG. 7C is a graphical representation of one embodiment of the arcuatepath crossing the chrome border of FIG. 7B including a plurality ofexcitation beam spots;

FIG. 7D is an illustrative example of one embodiment of reflected-beamintensity data from the plurality of excitation spots of FIG. 7C asplotted on a graph including slope values;

FIG. 7E is an illustrative example of one embodiment of slope values ofFIG. 7D plotted on a graph including a point associated with the bestplane of focus;

FIG. 8 is a simplified graphical representation of one embodiment of thescanner and auto-loader devices of FIG. 4;

FIG. 9 is a simplified graphical representation of one embodiment of thescanner and auto-loader devices of FIG. 8 with the cover for theauto-loader removed, showing one embodiment of an internal configurationincluding a cartridge magazine;

FIG. 10 is a graphical representation of one embodiment of the cartridgemagazine of FIG. 9;

FIG. 11 is a graphical representation of one embodiment of a transportassembly that includes reversible rollers;

FIG. 12 is a graphical representation of one embodiment of a probe arraycartridge and the reversible rollers of FIG. 11;

FIG. 13 is a graphical representation of one embodiment of the transportassembly of FIG. 11 including one embodiment of a cartridge magazinebase;

FIG. 14 is a graphical representation of one embodiment of the transportassembly of FIGS. 11 and 13 and the cartridge magazine of FIG. 10assembled together;

FIG. 15 is a graphical representation of one embodiment of a barcodelabel including a primary and secondary barcode;

FIG. 16 is a graphical representation of one embodiment of the cartridgetransport frame of FIG. 5 that includes a cartridge holder, pitch androll mechanisms, Y-stage, and a focus stage;

FIG. 17 is a graphical representation of one embodiment of the cartridgeholder and pitch and roll mechanisms of FIG. 16 including a pitch fingerand a roll finger;

FIG. 18 is a graphical representation of one embodiment of a four-barflexure focus stage guide;

FIG. 19 is a graphical representation of one embodiment of the four-barflexure focus stage guide of FIG. 18 assembled as a component of thefocus stage of FIG. 16;

FIG. 20A is a functional block diagram of one embodiment of calibrationexecutables of FIG. 6 including a calibration data generator, a radiusdistance generator, and an image correction correlator;

FIG. 20B is a functional block diagram of one embodiment of scannerfirmware executables of FIG. 6 including an image correction correlatorand an image acquirer;

FIG. 21 is a functional block diagram of one embodiment of thecalibration data generator of FIG. 20A;

FIG. 22 is a functional block diagram of one embodiment of a method forthe correction of image data using the calibration data table of FIG.20A;

FIG. 23 is a functional block diagram of one embodiment of a method forthe creation of a calibration data table;

FIG. 24 is a functional block diagram of one embodiment of the imagecorrection correlator of FIG. 20B including a corrected image generator;

FIG. 25 is a graphical representation of one embodiment of the scanningarm of FIG. 2 illustrating the potential difference between the radiusof the expected light path and the radius of the actual light path;

FIG. 26 is a graphical representation of one embodiment of data pointsfrom a scanning arc that are used to calculate the radius of the actuallight path of FIG. 25;

FIG. 27 is a functional block diagram of one embodiment of a method forthe calculation of the radius of the actual light path of FIG. 25;

FIG. 28 is a functional block diagram of one embodiment of the scannerfirmware interaction with linear encoder for control of the Y-Stagemotor of FIG. 16;

FIG. 29 is a functional block diagram of one embodiment of a method forlinear encoder control of Y-Stage motor of FIG. 16;

FIG. 30A is a graphical representation of one embodiment of acalibration tool including a rotational micrometer;

FIG. 30B is a graphical representation of one embodiment of thecalibration tool of FIG. 30A including a Y-axis micrometer and the galvoarm of FIG. 2.

DETAILED DESCRIPTION

The description below is designed to present specific embodiments andnot to be construed as limiting in any way. Also, reference will be madeto articles and patents to show general features that are incorporatedinto the present disclosure, but the invention is not limited by thesedescriptions. Many scanner designs may be used in order to provideexcitation and emission signals appropriate for the acquisition ofexperimental data derived from probe array 240.

In reference to the illustrative implementation of FIG. 1, the term“excitation beam” refers to light beams generated by lasers. However,excitation sources other than lasers may be used in alternativeimplementations. For example, other implementations may use lightemitting diodes, an incandescent light source, or any other light orother electromagnetic source of energy having a wavelength in theexcitation band of an excitable label, or capable of providingdetectable transmitted, reflective, or diffused radiation. Thus, theterm “excitation beam” is used broadly herein. The term “emission beam”also is used broadly herein. A variety of conventional scanners detectfluorescent or other emissions from labeled target molecules or othermaterial associated with biological probes. Other conventional scannersdetect transmitted, reflected, or scattered radiation from such targets.These processes are sometimes generally and collectively referred tohereafter for convenience simply as involving the detection of “emissionbeams.” Various detection schemes are employed depending on the type ofemissions and other factors. A typical scheme employs optical and otherelements to provide an excitation beam, such as from a laser, and toselectively collect the emission beams. Also generally included arevarious light-detector systems employing photodiodes, charge-coupleddevices, photomultiplier tubes, or similar or other conventional devicesto register the collected emission beams. For example, a scanning systemfor use with a fluorescently labeled target is described in U.S. Pat.Nos. 5,143,854 and 6,225,625, hereby incorporated by reference in theirentireties for all purposes. Other scanners or scanning systems aredescribed in U.S. Pat. Nos. 5,578,832; 5,631,734; 5,834,758; 5,936,324;5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,207,960;6,218,803; 6,252,236; 6,335,824; 6,490,533; 6,407,858; and 6,403,320;and in PCT Application PCT/US99/06097 (published as WO99/47964), each ofwhich also is hereby incorporated by reference in its entirety for allpurposes.

One embodiment of a scanning system as described above may serve avariety of functions. For example, some functions could include whatthose of ordinary skill in the related art refer to as expressionanalysis and/or genotyping. Genotyping could further include functionssuch as identification of single nucleotide polymorphisms, DNAsequencing, loss of heterozygosity identification, or transcriptomeanalysis. The previous examples are for the purposes of illustrationonly and it will be appreciated to those of ordinary skill in the artthat many additional functions may exist. An additional example of afunction could include diagnostic or other type of clinical uses. Forinstance, the diagnostic function could include the analysis of patientsamples for the purpose of identifying the existence or potential threatof health problems.

Additionally, a particularly embodiment of a scanning system asdescribed herein is constructed and arranged to accurately imagefeatures of a probe array that may include feature sizes of 18 μm, 10μm, or smaller in a dimension (such as the side of a square or side of arectangle). Accurately imaging very small feature sizes generallyrequires that tolerances for error associated with the hardware andsoftware elements of a scanning system be similarly small such as, forinstance, allowable position error may not exceed +/−1 pixel of anacquired image. Due to a variety of factors, multiple pixels aregenerally collected for every feature; for example, a range of 4-64pixels may be acquired for each feature depending upon feature size ofthe particular implementation of probe array 240.

Scanner Optics and Detectors 100: FIG. 1 is a simplified graphicalrepresentation of illustrative scanner optics and detectors (hereafter,simply “scanner optics”) 100. Although only one excitation source 120 isshown in the illustrated examples, any number of one or more excitationsources 120 may be used in alternative embodiments. In the presentexample, source 120 is a laser; in this embodiment a solid state, diodepumped, frequency doubled Nd: YAG (Neodymium-doped Yttrium AluminumGarnet) or YVO₄ laser producing green laser light having a wavelength of532 nm. Only one laser and accordingly, only one illumination beam isused during each scan. Further references herein to source 120 generallywill assume for illustrative purposes that they are lasers, but, asnoted, other types of sources, e.g., x-ray sources, may be used in otherimplementations. The Handbook of Biological Confocal Microscopy (JamesB. Pawley, ed.) (2.ed.; 1995; Plenum Press, NY), includes informationknown to those of ordinary skill in the art regarding the use of lasersand associated optics, is hereby incorporated herein by reference in itsentirety.

FIG. 1 further provides an illustrative example of the paths ofexcitation beam 135 and emission beam 152 and a plurality of opticalcomponents that comprise scanner optics 100. In the present example,excitation beam 135 is emitted from source 120 and is directed along anoptical path by one or more turning minors 124 toward a three-lens beamconditioner/expander 130. Turning mirrors are used in the optics moduleto provide the necessary adjustments to the optical path to allow foralignment of the excitation beam at the objective lens and to allow foralignment of the emission beam at the fluorescent detection module. Theturning mirrors also serve to “fold” the optical path into a morecompact size & shape to facilitate overall scanner packaging. The numberof turning mirrors may vary in different embodiments and may depend onthe requirements of the optical path. For instance, if the optical pathis required to turn a number of corners, such as 2 or more, then acorresponding number of turning mirrors 124 may be required to meet theoptical path requirements.

In some embodiments it may be desirable that excitation beam 135 has aknown diameter. Beam conditioner/expander 130 may provide one or moreoptical elements that adjust a beam diameter to a value that could, forinstance, include a diameter of 1.076 mm ±10%. For example, the one ormore optical elements could include a three-lens beam expander that mayincrease the diameter of excitation beam 135 to a desired value.Alternatively, the one or more optical elements may reduce the diameterof excitation beam 135 to a desired value. Additionally, the one or moreoptical elements of beam conditioner/expander 130 may farther conditionone or more properties of excitation beam 135 to provide other desirablecharacteristics, such as providing what those of ordinary skill in therelated art refer to as a plane wavefront to objective lens 145.Excitation beam 135 with the desirable characteristics may then exitbeam conditioner/expander 130 and continue along the optical path thatmay again be redirected by one or more turning minors 124 towardsexcitation filter 125.

Filter 125 may be used to remove light at wavelengths other thanexcitation wavelengths, and generally need not be included if, forexample, source 120 does not produce light at these extraneouswavelengths. However, it may be desirable in some applications to useinexpensive lasers and often it is cheaper to filter out-of-mode laseremissions than to design the laser to avoid producing such extraneousemissions. In some embodiments, filter 125 allows all or a substantialportion of light at the excitation wavelengths to pass through withoutaffecting other characteristics of excitation beam 135, such as thedesirable characteristics modified by beam conditioner/expander 130. Forexample, an excitation wavelength may include 532 nanometer (nm) thatmay excite one or more fluorophores including, for instance,R-phycoerythrin.

After exiting filter 125 excitation beam 135 may then be directed alongthe optical path to laser attenuator 133. Laser attenuator 133 mayprovide a means for adjusting the level of power of excitation beam 135.In some embodiments, attenuator 133 may, for instance, be comprised of avariable neutral density filter. Those of ordinary skill in the relatedart will appreciate that neutral density filters, such as absorptive,metallic, or other type of neutral density filter, may be used forreducing the amount of light that is allowed to pass through. The amountof light reduction may depend upon what is referred to as the density ofthe filter, for instance, as the density increases the amount of lightallowed to pass through decreases. The neutral density filter mayadditionally include a density gradient. For example, the presentlydescribed embodiment may include laser attenuator 133 that includes aneutral density filter with a density gradient. Attenuator 133, actingunder the control of scanner firmware executables 672 using one or moreparameters supplied by scanner control and analysis executables 572, mayuse a step motor that alters the position of the neutral density filterwith respect to the optical path. Attenuator 133 may make adjustments tothe level of laser power delivered to the probe array based, at least inpart, upon instructions from firmware executables 672. The adjustmentsmay be made by placing a specific density of the neutral density filtergradient, associated with a desired laser power, in the optical path.The neutral density filter thus reduces the amount of light allowed topass through to a desired level, as measured by laser power monitor 110,described in detail below.

Some embodiments may include one or more implementations of shutter 134.Some implementations may include positioning shutter 134 in one or morelocations within scanner 400, along the optical path such that shutter134 provides a means to block all laser light from reaching probe array240, and in some implementations additionally blocking all laser lightfrom reaching laser power monitor 110. Shutter 134 may use a variety ofmeans to completely block the light beam. For example shutter 134 mayuse a motor under the control of scanner firmware executables 672 toextend/retract a solid barrier that could be constructed of metal,plastic, or other appropriate material capable of blocking essentiallyall of the laser light beam, such as excitation beam 135. Shutter 134may be used for a variety of purposes such as, for example, for blockingall light from one or more photo detectors or monitors, includingemission detector 115 and laser power monitor 110. In the presentexample, blocking the light may be used for calibration methods such asmethods implemented by auto-zero executables 674 and/or calibrationexecutables and data 676 that measure and make adjustments to what isreferred to as the “dark current” or background noise of the photodetectors.

Components of scanner optics and detectors 100 placed in the opticalpath after elements such as attenuator 133 and/or shutter 134 mayinclude dichroic beam splitter 136. Those of ordinary skill in therelated art will appreciate that a dichroic beam splitter, also commonlyreferred to as a dichroic mirror, may include an optical element that ishighly reflective to light of a certain wavelength range, and allowtransmission of light through the beam splitter or mirror at one or moreother wavelength ranges. Alternatively, the beam splitter or mirror mayreflect a certain percentage of light at a particular wavelength andallow transmission of the remaining percentage. For example, dichroicbeam splitter 136 may direct most of the excitation beam, illustrated asexcitation beam 135′, along an optical path towards objective lens 145while allowing the small fractional portion of excitation beam 135 thatis not reflected to pass through beam splitter 136, illustrated in FIG.1 as partial excitation beam 137. For example, partial excitation beam137 passes through dichroic beam splitter 136 to laser power monitor 110for the purpose of measuring the power level of excitation beam 135 andproviding feedback to scanner firmware executables 672. Firmwareexecutables 672 may then makes adjustments, if necessary, to the powerlevel via laser attenuator 133.

Detector 110 may be any of a variety of conventional devices fordetecting partial excitation beam 137, such as a silicon detector forproviding an electrical signal representative of detected light, aphotodiode, a charge-coupled device, a photomultiplier tube, or anyother detection device for providing a signal indicative of detectedlight that is now available or that may be developed in the future. Asillustrated in FIG. 1, detector 110 generates excitation signal 194 thatrepresents the detected signal from partial excitation beam 137. Inaccordance with known techniques, the amplitude, phase, or othercharacteristic of excitation signal 194 is designed to vary in a knownor determinable fashion depending on the power of excitation beam 135.The term “power” in this context refers to the capability of beam 135 toevoke emissions. For example, the power of beam 135 typically may bemeasured in milliwatts of laser energy with respect to the illustratedexample in which the laser energy evokes a fluorescent signal. Thus,excitation signal 194 has values that represent the power of beam 135during particular times or time periods. Scanner firmware executables672 may receive signal 194 for evaluation and, as described above, ifnecessary make adjustments.

After reflection from beam splitter 136, excitation beam 135′ maycontinue along an optical path that is directed via periscope minor 138,turning mirror 140, and arm end turning mirror 142 to objective lens145. In the illustrated implementation minors 138, 140, and 142 may havethe same reflective properties as turning mirrors 124, and could, insome implementations, be used interchangeably with turning mirrors 124.As described in greater detail below in relation to FIGS. 2A and 2B,lens 145 in the illustrated implementation is a small, light-weight lenslocated on the end of an arm that is driven by a galvanometer around anaxis perpendicular to the plane represented by galvo rotation 149. Inone embodiment, lens 145 focuses excitation beam 135′ down to aspecified spot size at the best plane of focus that could, for instance,include a 3.5 μm spot size. Galvo rotation 149 results in objective lens145 moving in an arc over a substrate, illustrated in FIG. 2 as arcuatepath 250 that may also be referred to herein as a “scanning line”, uponwhich biological materials typically have been synthesized or have beendeposited. Arcuate path 250 may, for instance, move in a 36 degree arcover a substrate. Fluorophores associated with these biologicalmaterials emit emission beam 152 at characteristic wavelengths inaccordance with well-known principles. The term “fluorophore” commonlyrefers to a molecule that produces fluorescent light by energy transferfrom light, chemical, or other types of energy sources.

Emission beam 152 in the illustrated example follows the reverse opticalpath as described with respect to excitation beam 135 until reachingdichroic beam splitter 136. In accordance with well known techniques andprinciples, the characteristics of beam splitter 136 are selected sothat beam 152 (or a portion of it) passes through the minor rather thanbeing reflected. Emission beam 152 is then directed along a desiredoptical path to filter wheel 160.

In one embodiment, filter wheel 160 may be provided to filter outspectral components of emission beam 152 that are outside of theemission band of the fluorophore. The emission band is determined by thecharacteristic emission frequencies of those fluorophores that areresponsive to the frequency of excitation beam 135. Thus, for example,excitation beam 135 from source 120, which is illustratively assumed tohave a wavelength of 532 nanometers, excites certain fluorophores to amuch greater degree than others. The characteristic emission wavelengthof a first illustrative fluorophore (not shown in FIG. 1) when excitedby beam 135 may be assumed to be 551 nanometers. Emission beam 152 inthis example typically will also include wavelengths above and below 551nanometers in accordance with distributions that are known to those ofordinary skill in the relevant art. The result may include filteredemission beam 154 that is a representation of emission beam 152 that hasbeen filtered by a desired filter of filter wheel 160.

In some implementations filter wheel 160 is capable of holding aplurality of filters that each could be tuned to different wavelengthscorresponding to the emission spectra from different fluorophores.Filter wheel 160 may include a mechanism for turning the wheel toposition a desired filter in the optical path of emission beam 152. Themechanism may include a motor or some other device for turning that maybe responsive to instructions from scanner firmware executables 672. Forexample, biological probe array experiments could be carried out on thesame probe array where a plurality of fluorophores with differentemission spectra are used that could be excited by a single or multiplelasers. Dyes that use the same excitation wavelengths but have differingemission spectral properties could be produced by methods such as thoseknown to those in the aft as fluorescent resonant energy transfer (FRET)where two fluorophores are present in the same molecule. The emissionwavelength of one fluorophore overlaps the excitation wavelength of thesecond fluorophore and results in the emission of a wavelength from thesecond fluorophore that is atypical of the class of fluorophores thatuse that excitation wavelength. Thus by using one excitation beam it ispossible to obtain distinctly different emissions so that differentfeatures of a probe array could be labeled in a single experiment.

In the present example the probe array could be scanned using a filterof one wavelength, then one or more additional scans could be performedthat each correspond to a particular fluorophore and filter pair.Scanner control and analysis executables 572 could then process the dataso that the user could be presented with a single image or other formatfor data analysis. In other implementations, multiple excitation sources120 (or one or more adjustable-wavelength excitation sources) andcorresponding multiple optical elements in optical paths similar to theillustrated one could be employed for simultaneous scans at multiplewavelengths. Other examples of scanner systems that utilize multipleemission wavelengths are described in U.S. patent application Ser. No.09/683,216 titled “System, Method, and Product For Dynamic NoiseReduction in Scanning of Biological Materials”, filed Dec. 3, 2001; U.S.patent application Ser. No. 09/683,217 titled “System, Method, andProduct for Pixel Clocking in Scanning of Biological Materials”, filedDec. 3, 2001; and U.S. patent application Ser. No. 09/683,219 titled“System, Method, and Product for Symmetrical Filtering in Scanning ofBiological Materials”, filed Dec. 3, 2001 each of which are herebyincorporated by reference in their entireties for all purposes.

In accordance with techniques well known to those of ordinary skill inthe relevant arts, including that of confocal microscopy, beam 154 maybe focused by various optical elements such as lens 165 and passedthrough illustrative pinhole 167, aperture, or other element. Inaccordance with known techniques, pinhole 167 is positioned such that itrejects light from focal planes other than the plane of focus ofobjective lens 145 (i.e., out-of-focus light), and thus increases theresolution of resulting images. After passing through pinhole 167, theportion of filtered emission beam 154 that corresponds to the plane offocus, represented as filtered emission beam 154′, continues along adesired optical path and impinges upon emission detector 115.

Similar to excitation detector 110, emission detector 115 may be asilicon detector for providing an electrical signal representative ofdetected light, or it may be a photodiode, a charge-coupled device, aphotomultiplier tube, or any other detection device that is nowavailable or that may be developed in the future for providing a signalindicative of detected light. Detector 115 generates emission signal 192and/or calibration signal 196 that represents filtered emission beam154′ in the manner noted above with respect to the generation ofexcitation signal 194 by detector 110. Emission signal 192 andexcitation signal 194 are provided to scanner firmware executables 672for processing, as described below in relation to FIG. 5.

In the same or other implementations, one or more additional dichroicmirrors may be placed in the optical path of emission beam 152represented in FIG. 1 as dichroic mirror 170. Each of the one or moremirrors 170 could be tuned to select specific wavelengths of emissionbeam 152 to reflect. The reflected beam of the selected wavelength isrepresented in FIG. 1 as selected emission beam 175. Selected emissionbeam 175 could, in some implementations, be directed to additionaldetection devices that may be the same type of detection device asemission detectors 110 or 115 represented as selected beam detectiondevice 180. Device 180 may also be associated with implementations ofthe above described optical elements, such as filter wheel 160, lens165, and pinhole 167. For example, biological probe array experimentscould be carried out on the same probe array where a plurality offluorophores with different emission spectra is used. Each of thefluorophores may have a distinct emission spectra that has an associatedimplementation of mirror 170 tuned to reflect the distinct emissionspectra and allow all other spectra to pass through as previouslydescribed with respect to beam splitter 136. In the present example,each of mirrors 170 has an associated filter wheel 160, lens 165, andpinhole 167, as well as detector 180. Each of detectors 180 associatedgenerates a signal similar to emission signal 192 that is specific tothe detected spectra reflected by mirror 170. Each of signals 192 maythen be forwarded to scanner firmware and additionally sent to scannercontrol an analysis executables for further processing. Additionally, inthe present example a single scan may be performed where thefluorophores are excited by the excitation beam and the distinctemission spectra associated with the fluorophores could be selected forby dichroic mirrors 170, as opposed to the situation where a separatescan may need to be performed for each fluorophore using a singleimplementation of detector 115 and associated optical elements. Benefitsfrom this configuration could include a reduced amount of what isreferred to by those in the art as photobleaching. Photobleaching is acharacteristic of fluorescent emissions according to which the amount oftime that a fluorophore is exposed to the excitation light correspondsto a reduction in emission intensity until it is reduced to a value thatmay be zero. Additionally, the implementation described in the examplemay require less time to scan an array using multiple fluorophoresbecause it performs a single scan as opposed to multiple scans.

A method for aligning the optical paths within scanner 400 such as,excitation beam 135 and emission beam 152 may include the use of aplurality of optical barriers. In a similar fashion as with respect toshutter 134, the plurality of optical barriers could be constructed ofmetal, plastic, or other appropriate material capable of blockingessentially all of the laser light beam. Each of the optical barriersmay include a pinhole type aperture that allows for a beam of light topass completely through the optical barrier, and where each of thepinhole type apertures is slightly larger than either of beams 135 or152. Each pinhole type aperture may be located at a known position inthe optical barrier such as, for instance, in the center of the opticalbarrier, although it is not necessary that the known position of thepinhole type aperture be the same position for each optical barrier. Themethod may include placing each optical barrier with associated pinholetype aperture in successive positions in the optimal desired opticalpath within scanner 400. In one embodiment of the method, the first stepof aligning may include placing a first optical barrier with anassociated pinhole type aperture in a fixed position close to laser 120,or alternatively close to a turning mirror, such as turning mirror 124.The pinhole type aperture must be placed in the optical path that thebeam must travel through to be properly aligned. Adjustment of theoptical path of the beam may be required if the beam does not passthrough the pinhole type aperture. Adjustment may be accomplished eitherby adjusting the aim and/or position of laser 120, and/or adjustments toone or more turning mirrors may be made, until the beam passes throughthe pinhole type aperture. The method may be repeated with opticalbarriers being placed at successive positions within the optical path,where each successive position is farther away from laser 120 than thelast position, until the entire optical path has been aligned. In thepresently described method, the fixed positions may be located so thateach optical barrier may be supported by some fixed, rigid structurewithin scanner 400. Additionally, the number of optical barriers mayvary as needed by the complexity of the light path within the scanner.

FIG. 2A illustrates an example of a perspective view of a simplifiedrepresentation of a scanning arm portion of scanner optics 100 inaccordance with this particular, non-limiting, implementation. Arm 200moves in arcs around axis 210, which is perpendicular to the plane ofgalvo rotation 149. A position transducer may also be associated witharm 200. A possible benefit of this construction may be that theposition of the oscillating arm itself is determined at each instant ofdata collection. Exact position data from the position transducer may beused for the construction of linearity correction tables and used forimage correction methods that will be described in greater detail belowin reference to calibration and image-correction methods. The positiontransducer, in accordance with any of a variety of known techniques,provides an electrical signal indicative of the radial position of arm200. Certain non-limiting implementations of position transducers forgalvanometer-driven scanners are described in U.S. Pat. No. 6,218,803that is hereby incorporated by reference in its entirety for allpurposes.

Arm 200 is shown in alternative position 200′ and 200″ as it moves backand forth in scanning arcs about axis 210. Excitation beams 135 passthrough objective lens 145 on the end of arm 200 and excite fluorophoresthat may be contained in hybridized probe-target pairs in features 230on a substrate of probe array 240, as further described below. Thearcuate path of excitation beams 135 over probe array 240 isschematically shown for illustrative purposes as path 250. Emissionbeams 152 pass up through objective lens 145 as noted above. Probe array240 of this example is disposed on translation stage 242 that is movedin direction 244 so that arcuate path 250 repeatedly crosses the planeof probe array 240. The example of stage 242 is presented for thepurposes of illustration only, and will be understood that otherembodiments exist such as, for instance, one or more elements ofcartridge transport frame 505 that will be discussed in detail below. Asis evident, the resulting coverage of excitation beams 135 over theplane of probe array 240 in this implementation is therefore determinedby the footprint of beam, the speed of movement in direction 244, andthe speed of the scan. FIG. 2B is a top planar view of arm 200 withobjective lens 145 scanning features 230 on probe array 240 astranslation stage 242 is moved under path 250. As shown in FIG. 2B,arcuate path 250 of this example is such that arm 200 has a radialdisplacement of θ in each direction from an axis parallel to direction244. For convenience of reference below, a direction 243 perpendicularto direction 244 is also shown in FIG. 2B. For illustrative purposes,direction 243 may hereafter be referred to herein as the “x” direction,and direction 244 as the “y” direction.

In this implementation, the scan arm moves through a constant arcregardless of the array being scanned. As a result, the lens traversesthe same distance over the array, and accordingly, the same number ofpixels, regardless of where over the array the lens is traveling or thefluorescent material that may be present in the array. In addition, thetime it takes in this implementation for the lens to complete one passand return back to a starting position is the same, for example in onepossible implementation 1/50th of a second, regardless of the arraybeing scanned or the fluorescent materials that may be present.Alternatively, some implementations may include the scan arm moving inan arc that is dependant upon the size of probe array 240. For example,the width of the arc for a particular probe array may be stored inscanner parameter data 677 that may be used by scanner firmwareexecutables 672 to define the size of the arc.

Further details of confocal, galvanometer-driven, arcuate, laserscanning instruments suitable for detecting fluorescent emissions areprovided in PCT Application PCT/US99/06097 (published as WO99/47964) andin U.S. Pat. Nos. 6,185,030; 6,201,639; and 6,225,625, all of which havebeen incorporated by reference above.

Probe Array 240: Various techniques and technologies may be used forsynthesizing dense arrays of biological materials on or in a substrateor support. For example, Affymetrix GeneChip® arrays are synthesized inaccordance with techniques sometimes referred to as VLSIPS™ (Very LargeScale Immobilized Polymer Synthesis) technologies. Some aspects ofVLSIPS™ and other microarray and polymer (including protein) arraymanufacturing methods and techniques have been described in U.S. patentSer. No. 09/536,841, International Publication No. WO 00/58516; U.S.Pat. Nos. 5,143,854, 5,242,974, 5,252,743, 5,324,633, 5,445,934,5,744,305, 5,384,261, 5,405,783, 5,424,186, 5,451,683, 5,482,867,5,491,074, 5,527,681, 5,550,215, 5,571,639, 5,578,832, 5,593,839,5,599,695, 5,624,711, 5,631,734, 5,795,716, 5,831,070, 5,837,832,5,856,101, 5,858,659, 5,936,324, 5,968,740, 5,974,164, 5,981,185,5,981,956, 6,025,601, 6,033,860, 6,040,193, 6,090,555, 6,136,269,6,269,846, 6,022,963, 6,083,697, 6,291,183, 6,309,831 and 6,428,752; andin PCT Applications Nos. PCT/US99/00730 (International Publication No.WO 99/36760) and PCT/US01/04285, which are all incorporated herein byreference in their entireties for all purposes.

Patents that describe synthesis techniques in specific embodimentsinclude U.S. Pat. Nos. 6,486,287, 6,147,205, 6,262,216, 6,310,189,5,889,165, 5,959,098, and 5,412,087, all hereby incorporated byreference in their entireties for all purposes. Nucleic acid arrays aredescribed in many of the above patents, but the same techniquesgenerally may be applied to polypeptide arrays or other types of probearrays.

Generally speaking, an “array” typically includes a collection ofmolecules that can be prepared either synthetically or biosynthetically.The molecules in the array may be identical, they may be duplicative,and/or they may be different from each other. The array may assume avariety of formats, e.g., libraries of soluble molecules; libraries ofcompounds tethered to resin beads, silica chips, or other solidsupports; and other formats.

The terms “solid support,” “support,” and “substrate” may in somecontexts be used interchangeably and may refer to a material or group ofmaterials having a rigid or semi-rigid surface or surfaces, which maybe, or have coverings that are, porous or not and that may provide twoor three dimensions for the securing of probes. In many embodiments, atleast one surface of a solid support will be substantially flat,although in some embodiments it may be desirable to physically separatesynthesis regions for different compounds with, for example, wells,raised regions, pins, etched trenches or wells, or other separationmembers or elements. In some embodiments, the solid support(s) may takethe form of beads, resins, gels, microspheres, or other materials and/orgeometric configurations.

Generally speaking, a “probe” typically is a molecule that can berecognized by a particular target. To ensure proper interpretation ofthe term “probe” as used herein, it is noted that contradictoryconventions exist in the relevant literature. The word “probe” is usedin some contexts to refer not to the biological material that issynthesized on a substrate or deposited on a slide, as described above,but to what is referred to herein as the “target.”

A target is a molecule that has an affinity for a given probe. Targetsmay be naturally-occurring or man-made molecules. Also, they can beemployed in their unaltered state or as aggregates with other species.The samples or targets are processed so that, typically, they arespatially associated with certain probes in the probe array. Forexample, one or more tagged targets may be distributed over the probearray.

Targets may be attached, covalently or noncovalently, to a bindingmember, either directly or via a specific binding substance. Examples oftargets that can be employed in accordance with this invention include,but are not restricted to, antibodies, cell membrane receptors,monoclonal antibodies and antisera reactive with specific antigenicdeterminants (such as on viruses, cells or other materials), drugs,oligonucleotides, nucleic acids, peptides, cofactors, lectins, sugars,polysaccharides, cells, cellular membranes, and organelles. Targets aresometimes referred to in the art as anti-probes. As the term target isused herein, no difference in meaning is intended. Typically, a“probe-target pair” is formed when two macromolecules have combinedthrough molecular recognition to form a complex.

The probes of the arrays in some implementations comprise nucleic acidsthat are synthesized by methods including the steps of activatingregions of a substrate and then contacting the substrate with a selectedmonomer solution. The term “monomer” generally refers to any member of aset of molecules that can be joined together to form an oligomer orpolymer. The set of monomers useful in the present invention includes,but is not restricted to, for the example of (poly) peptide synthesis,the set of L-amino acids, D-amino acids, or synthetic amino acids. Asused herein, “monomer” refers to any member of a basis set for synthesisof an oligomer. For example, dimers of L-amino acids form a basis set of400 “monomers” for synthesis of polypeptides. Different basis sets ofmonomers may be used at successive steps in the synthesis of a polymer.The term “monomer” also refers to a chemical subunit that can becombined with a different chemical subunit to form a compound largerthan either subunit alone. In addition, the terms “biopolymer” and“biological polymer” generally refer to repeating units of biological orchemical moieties. Representative biopolymers include, but are notlimited to, nucleic acids, oligonucleotides, amino acids, proteins,peptides, hormones, oligosaccharides, lipids, glycolipids,lipopolysaccharides, phospholipids, synthetic analogues of theforegoing, including, but not limited to, inverted nucleotides, peptidenucleic acids, Meta-DNA, and combinations of the above. “Biopolymersynthesis” is intended to encompass the synthetic production, bothorganic and inorganic, of a biopolymer. Related to the term “biopolymer”is the term “biomonomer” that generally refers to a single unit ofbiopolymer, or a single unit that is not part of a biopolymer. Thus, forexample, a nucleotide is a biomonomer within an oligonucleotidebiopolymer, and an amino acid is a biomonomer within a protein orpeptide biopolymer; avidin, biotin, antibodies, antibody fragments,etc., for example, are also biomonomers.

As used herein, nucleic acids may include any polymer or oligomer ofnucleosides or nucleotides (polynucleotides or oligonucleotides) thatinclude pyrimidine and/or purine bases, preferably cytosine, thymine,and uracil, and adenine and guanine, respectively. An “oligonucleotide”or “polynucleotide” is a nucleic acid ranging from at least 2,preferable at least 8, and more preferably at least 20 nucleotides inlength or a compound that specifically hybridizes to a polynucleotide.Polynucleotides of the present invention include sequences ofdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA), which may beisolated from natural sources, recombinantly produced or artificiallysynthesized and mimetics thereof. A further example of a polynucleotidein accordance with the present invention may be peptide nucleic acid(PNA) in which the constituent bases are joined by peptides bonds ratherthan phosphodiester linkage, as described in Nielsen et al., Science254:1497-1500 (1991); Nielsen, Curr. Opin. Biotechnol., 10:71-75 (1999),both of which are hereby incorporated by reference herein. The inventionalso encompasses situations in which there is a nontraditional basepairing such as Hoogsteen base pairing that has been identified incertain tRNA molecules and postulated to exist in a triple helix.“Polynucleotide” and “oligonucleotide” may be used interchangeably inthis application.

Additionally, nucleic acids according to the present invention mayinclude any polymer or oligomer of pyrimidine and purine bases,preferably cytosine (C), thymine (T), and uracil (U), and adenine (A)and guanine (G), respectively. See Albert L. Lehninger, PRINCIPLES OFBIOCHEMISTRY, at 793-800 (Worth Pub. 1982). Indeed, the presentinvention contemplates any deoxyribonucleotide, ribonucleotide orpeptide nucleic acid component, and any chemical variants thereof, suchas methylated, hydroxymethylated or glucosylated forms of these bases,and the like. The polymers or oligomers may be heterogeneous orhomogeneous in composition, and may be isolated from naturally occurringsources or may be artificially or synthetically produced. In addition,the nucleic acids may be deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), or a mixture thereof, and may exist permanently or transitionallyin single-stranded or double-stranded form, including homoduplex,heteroduplex, and hybrid states.

As noted, a nucleic acid library or array typically is an intentionallycreated collection of nucleic acids that can be prepared eithersynthetically or biosynthetically in a variety of different formats(e.g., libraries of soluble molecules; and libraries of oligonucleotidestethered to resin beads, silica chips, or other solid supports).Additionally, the term “array” is meant to include those libraries ofnucleic acids that can be prepared by spotting nucleic acids ofessentially any length (e.g., from 1 to about 1000 nucleotide monomersin length) onto a substrate. The term “nucleic acid” as used hereinrefers to a polymeric form of nucleotides of any length, eitherribonucleotides, deoxyribonucleotides or peptide nucleic acids (PNAs),that comprise purine and pyrimidine bases, or other natural, chemicallyor biochemically modified, non-natural, or derivatized nucleotide bases.The backbone of the polynucleotide can comprise sugars and phosphategroups, as may typically be found in RNA or DNA, or modified orsubstituted sugar or phosphate groups. A polynucleotide may comprisemodified nucleotides, such as methylated nucleotides and nucleotideanalogs. The sequence of nucleotides may be interrupted bynon-nucleotide components. Thus the terms nucleoside, nucleotide,deoxynucleoside and deoxynucleotide generally include analogs such asthose described herein. These analogs are those molecules having somestructural features in common with a naturally occurring nucleoside ornucleotide such that when incorporated into a nucleic acid oroligonucleotide sequence, they allow hybridization with a naturallyoccurring nucleic acid sequence in solution. Typically, these analogsare derived from naturally occurring nucleosides and nucleotides byreplacing and/or modifying the base, the ribose or the phosphodiestermoiety. The changes can be tailor made to stabilize or destabilizehybrid formation or enhance the specificity of hybridization with acomplementary nucleic acid sequence as desired. Nucleic acid arrays thatare useful in the present invention include those that are commerciallyavailable from Affymetrix, Inc. of Santa Clara, Calif., under theregistered trademark “GeneChip®.” Example arrays are shown on thewebsite at affymetrix.com.

In some embodiments, a probe may be surface immobilized. Examples ofprobes that can be investigated in accordance with this inventioninclude, but are not restricted to, agonists and antagonists for cellmembrane receptors, toxins and venoms, viral epitopes, hormones (e.g.,opioid peptides, steroids, etc.), hormone receptors, peptides, enzymes,enzyme substrates, cofactors, drugs, lectins, sugars, oligonucleotides,nucleic acids, oligosaccharides, proteins, and monoclonal antibodies. Asnon-limiting examples, a probe may refer to a nucleic acid, such as anoligonucleotide, capable of binding to a target nucleic acid ofcomplementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation. A probe may include natural (i.e. A, G, U, C, or T) ormodified bases (7-deazaguanosine, inosine, etc.). In addition, the basesin probes may be joined by a linkage other than a phosphodiester bond,so long as the bond does not interfere with hybridization. Thus, probesmay be peptide nucleic acids in which the constituent bases are joinedby peptide bonds rather than phosphodiester linkages. Other examples ofprobes include antibodies used to detect peptides or other molecules, orany ligands for detecting its binding partners. Probes of otherbiological materials, such as peptides or polysaccharides asnon-limiting examples, may also be formed. For more details regardingpossible implementations, see U.S. Pat. No. 6,156,501, herebyincorporated by reference herein in its entirety for all purposes. Whenreferring to targets or probes as nucleic acids, it should be understoodthat these are illustrative embodiments that are not to limit theinvention in any way.

Furthermore, to avoid confusion, the term “probe” is used broadly hereinto refer, for example, to probes such as those synthesized according tothe VLSIPS™ technology; the biological materials deposited so as tocreate spotted arrays; and materials synthesized, deposited, orpositioned to form arrays according to other current or futuretechnologies. Thus, microarrays formed in accordance with any of thesetechnologies may be referred to generally and collectively hereafter forconvenience as “probe arrays.” Moreover, the term “probe” is not limitedto probes immobilized in array format. Rather, the functions and methodsdescribed herein may also be employed with respect to other parallelassay devices. For example, these functions and methods may be appliedwith respect to probe-set identifiers that identify probes immobilizedon or in beads, optical fibers, or other substrates or media.

In accordance with some implementations, some targets hybridize withprobes and remain at the probe locations, while non-hybridized targetsare washed away. These hybridized targets, with their tags or labels,are thus spatially associated with the probes. The term “hybridization”refers to the process in which two single-stranded polynucleotides bindnon-covalently to form a stable double-stranded polynucleotide. The term“hybridization” may also refer to triple-stranded hybridization, whichis theoretically possible. The resulting (usually) double-strandedpolynucleotide is a “hybrid.” The proportion of the population ofpolynucleotides that forms stable hybrids is referred to herein as the“degree of hybridization.” Hybridization probes usually are nucleicacids (such as oligonucleotides) capable of binding in a base-specificmanner to a complementary strand of nucleic acid. Such probes includepeptide nucleic acids, as described in Nielsen et al., Science254:1497-1500 (1991) or Nielsen Curr. Opin. Biotechnol., 10:71-75 (1999)(both of which are hereby incorporated herein by reference), and othernucleic acid analogs and nucleic acid mimetics. The hybridized probe andtarget may sometimes be referred to as a probe-target pair. Detection ofthese pairs can serve a variety of purposes, such as to determinewhether a target nucleic acid has a nucleotide sequence identical to ordifferent from a specific reference sequence. See, for example, U.S.Pat. No. 5,837,832, referred to and incorporated above. Other usesinclude gene expression monitoring and evaluation (see, e.g., U.S. Pat.No. 5,800,992 to Fodor, et al.; U.S. Pat. No. 6,040,138 to Lockhart, etal.; and International App. No. PCT/US98/15151, published as WO99/05323,to Balaban, et al.), genotyping (U.S. Pat. No. 5,856,092 to Dale, etal.), or other detection of nucleic acids. The '992, '138, and '092patents, and publication WO99/05323, are incorporated by referenceherein in their entireties for all purposes.

The present invention also contemplates signal detection ofhybridization between probes and targets in certain embodiments. SeeU.S. Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,936,324; 5,981,956;6,025,601 incorporated above, and U.S. Pat. Nos. 5,834,758, 6,141,096;6,185,030; 6,201,639; 6,218,803; and 6,225,625, U.S. Patent application60/364,731, and PCT Application PCT/US99/06097 (published asWO99/47964), each of which also is hereby incorporated by reference inits entirety for all purposes.

A system and method for efficiently synthesizing probe arrays usingmasks is described in U.S. patent application Ser. No. 09/824,931, filedApr. 3, 2001, that is hereby incorporated by reference herein in itsentirety for all purposes. A system and method for a rapid and flexiblemicroarray manufacturing and online ordering system is described in U.S.patent application Ser. No. 10/065,868 filed Nov. 26, 2002, that also ishereby incorporated herein by reference in its entirety for allpurposes. Systems and methods for optical photolithography without masksare described in U.S. Pat. No. 6,271,957 and in U.S. patent applicationSer. No. 09/683,374 filed Dec. 19, 2001, both of which are herebyincorporated by reference herein in their entireties for all purposes.

As noted, various techniques exist for depositing probes on a substrateor support. For example, “spotted arrays” are commercially fabricated,typically on microscope slides. These arrays consist of liquid spotscontaining biological material of potentially varying compositions andconcentrations. For instance, a spot in the array may include a fewstrands of short oligonucleotides in a water solution, or it may includea high concentration of long strands of complex proteins. TheAffymetrix® 417™ Arrayer and 427™ Arrayer are devices that depositdensely packed arrays of biological materials on microscope slides inaccordance with these techniques. Aspects of these and other spotarrayers are described in U.S. Pat. Nos. 6,040,193 and 6,136,269 and inPCT Application No. PCT/US99/00730 (International Publication Number WO99/36760) incorporated above and in U.S. patent application Ser. No.09/683,298 hereby incorporated by reference in its entirety for allpurposes. Other techniques for generating spotted arrays also exist. Forexample, U.S. Pat. No. 6,040,193 to Winkler, et al. is directed toprocesses for dispensing drops to generate spotted arrays. The '193patent, and U.S. Pat. No. 5,885,837 to Winkler, also describe the use ofmicro-channels or micro-grooves on a substrate, or on a block placed ona substrate, to synthesize arrays of biological materials. These patentsfurther describe separating reactive regions of a substrate from eachother by inert regions and spotting on the reactive regions. The '193and '837 patents are hereby incorporated by reference in theirentireties. Another technique is based on ejecting jets of biologicalmaterial to form a spotted array. Other implementations of the jettingtechnique may use devices such as syringes or piezo electric pumps topropel the biological material. It will be understood that the foregoingare non-limiting examples of techniques for synthesizing, depositing, orpositioning biological material onto or within a substrate. For example,although a planar array surface is desirable in some implementations ofthe foregoing, a probe array may be fabricated on a surface, or in athree-dimensional space, of virtually any shape or even a multiplicityof shapes and/or surfaces. Arrays may comprise probes synthesized ordeposited on beads, fibers such as fiber optics, glass, silicon, silicaor any other appropriate substrate, see U.S. Pat. No. 5,800,992 referredto and incorporated above and U.S. Pat. Nos. 5,770,358, 5,789,162,5,708,153 and 6,361,947 all of which are hereby incorporated in theirentireties for all purposes. Arrays may be packaged in such a manner asto allow for diagnostics or other manipulation in an all inclusivedevice, see for example, U.S. Pat. Nos. 5,856,174 and 5,922,591 herebyincorporated in their entireties by reference for all purposes.

Probes typically are able to detect the expression of correspondinggenes or EST's by detecting the presence or abundance of mRNAtranscripts present in the target. This detection may, in turn, beaccomplished in some implementations by detecting labeled cRNA that isderived from cDNA derived from the mRNA in the target.

The terms “mRNA” and “mRNA transcripts” as used herein, include, but notlimited to pre-mRNA transcript(s), transcript processing intermediates,mature mRNA(s) ready for translation and transcripts of the gene orgenes, or nucleic acids derived from the mRNA transcript(s). Thus, mRNAderived samples include, but are not limited to, mRNA transcripts of thegene or genes, cDNA reverse transcribed from the mRNA, cRNA transcribedfrom the cDNA, DNA amplified from the genes, RNA transcribed fromamplified DNA, and the like.

In general, a group of probes, sometimes referred to as a probe set,contains sub-sequences in unique regions of the transcripts and does notcorrespond to a full gene sequence. Further details regarding the designand use of probes and probe sets are provided in PCT Application SerialNo. PCT/US 01/02316, filed Jan. 24, 2001 incorporated above; and in U.S.Pat. No. 6,188,783 and in U.S. patent application Ser. No. 09/721,042,filed on Nov. 21, 2000, Ser. No. 09/718,295, filed on Nov. 21, 2000,Ser. No. 09/745,965, filed on Dec. 21, 2000, and Ser. No. 09/764,324,filed on Jan. 16, 2001, all of which patent and patent applications arehereby incorporated herein by reference in their entireties for allpurposes.

LIMS Server 320: FIGS. 3 and 4 show a typical configuration of a servercomputer connected to a workstation computer via a network. Forconvenience, the server computer is referred to herein as LIMS server320, although this computer may carry out a variety of functions inaddition to those described below with respect to the implementations ofAffymetrix® LIMS, Affymetrix® AIMS, Affymetrix® GDAC, and Affymetrix®LIMS-SDK software applications. Moreover, in some implementations anyfunction ascribed to LIMS server 320 may be carried out by one or moreother computers, and/or the functions may be performed in parallel by agroup of computers. Network 325 may include a local area network, a widearea network, the Internet, another network, or any combination thereof.

An illustrative embodiment of LIMS server 320 is shown in greater detailin FIG. 4. Typically, LIMS server 320 is a network-server class ofcomputer designed for servicing a number of workstations or othercomputer platforms over a network. However, server 320 may be any of avariety of types of general-purpose computers such as a personalcomputer, workstation, main frame computer, or other computer platformnow or later developed. Server 320 typically includes known componentssuch as a processor 405, an operating system 410, a system memory 420,memory storage devices 425, and input-output controllers 430. It will beunderstood by those skilled in the relevant art that there are manypossible configurations of the components of server 320 and that somecomponents that may typically be included are not shown, such as cachememory, a data backup unit, and many other devices. Similarly, manyhardware and associated software or firmware components that may beimplemented in a network server are not shown in FIG. 4. For example,components to implement one or more firewalls to protect data andapplications, uninterruptible power supplies, LAN switches, web-serverrouting software, and many other components are not shown. Those ofordinary skill in the art will readily appreciate how these and otherconventional components may be implemented.

Processor 405 may include multiple processors; e.g., multiple IntelXeon® 700 MHz processors. As further examples, processor 405 may includeone or more of a variety of other commercially available processors suchas Pentium® processors from Intel, SPARC® processors made by SunMicrosystems, or other processors that are or will become available.Processor 405 executes operating system 410, which may be, for example,a Windows®-type operating system (such as Windows® 2000 with SP 1,Windows NT® 4.0 with SP6a) from the Microsoft Corporation; the Solarisoperating system from Sun Microsystems, the Tru64 Unix from Compaq,other Unix® or Linux-type operating systems available from many vendors;another or a future operating system; or some combination thereof.Operating system 410 interfaces with firmware, such as scanner firmware510, and hardware in a well-known manner. Operating system 410 alsofacilitates processor 405 in coordinating and executing the functions ofvarious computer programs that may be written in a variety ofprogramming languages. Operating system 410, typically in cooperationwith processor 405, coordinates and executes functions of the othercomponents of server 320. Operating system 410 also provides scheduling,input-output control, file and data management, memory management, andcommunication control and related services, all in accordance with knowntechniques.

System memory 420 may be any of a variety of known or future memorystorage devices. Examples include any commonly available random accessmemory (RAM), magnetic medium such as a resident hard disk or tape, anoptical medium such as a read and write compact disc, or other memorystorage device. Memory storage device 425 may be any of a variety ofknown or future devices, including a compact disk drive, a tape drive, aremovable hard disk drive, or a diskette drive. Such types of memorystorage device 425 typically read from, and/or write to, a programstorage medium (not shown) such as, respectively, a compact disk,magnetic tape, removable hard disk, or floppy diskette. Any of theseprogram storage media, or others now in use or that may later bedeveloped, may be considered a computer program product. As will beappreciated, these program storage media typically store a computersoftware program and/or data. Computer software programs, also calledcomputer control logic, typically are stored in system memory 420 and/orthe program storage device used in conjunction with memory storagedevice 425.

In some embodiments, a computer program product is described comprisinga computer usable medium having control logic (computer softwareprogram, including program code) stored therein. The control logic, whenexecuted by processor 405, causes processor 405 to perform functionsdescribed herein. In other embodiments, some functions are implementedprimarily in hardware using, for example, a hardware state machine.Implementation of the hardware state machine so as to perform thefunctions described herein will be apparent to those skilled in therelevant arts.

Input-output controllers 430 could include any of a variety of knowndevices for accepting and processing information from a user, whether ahuman or a machine, whether local or remote. Such devices include, forexample, modem cards, network interface cards, sound cards, or othertypes of controllers for any of a variety of known input or outputdevices. In the illustrated embodiment, the functional elements ofserver 320 communicate with each other via system bus 404. Some of thesecommunications may be accomplished in alternative embodiments usingnetwork or other types of remote communications.

As will be evident to those skilled in the relevant art, LIMS serverapplication 480, as well as LIMS Objects 490 including LIMS servers 492and LIMS API's 494 (described below), if implemented in software, may beloaded into system memory 420 and/or memory storage device 425 throughone of input devices 402. LIMS server application 480 as loaded intosystem memory 420 is shown in FIG. 12 as LIMS server applicationexecutables 480A. Similarly, objects 490 are shown as LIMS serverexecutables 492A and LIMS API object type libraries 494A after they havebeen loaded into system memory 420. All or portions of these loadedelements may also reside in a read-only memory or similar device ofmemory storage device 425, such devices not requiring that the elementsfirst be loaded through input devices 402. It will be understood bythose skilled in the relevant art that any of the loaded elements, orportions of them, may be loaded by processor 405 in a known manner intosystem memory 420, or cache memory (not shown), or both, as advantageousfor execution.

LIMS Server Application 480: Details regarding the operations ofillustrative implementations of application 480 are provided in U.S.patent application Ser. Nos. 09/682,098; 10/219,882; and Attorney DocketNumber 3348.7 (entitled System and Method for Programmatic Access toBiological Probe Array Data, filed Feb. 19, 2003), each of which ishereby incorporated by reference herein in its entirety for allpurposes. It will be understood that the particular LIMS implementationdescribed in this patent application is illustrative only, and that manyother implementations may be used with LIMS objects 490 and otheraspects of the present or alternative embodiments.

Application 480, and other software applications referred to herein, maybe implemented using Microsoft® Visual C++ or any of a variety of otherprogramming languages. For example, applications may also be written inJava, C++, Visual Basic, any other high-level or low-level programminglanguage, or any combination thereof. As noted, certain implementationsmay be illustrated herein with respect to a particular, non-limiting,implementation of application 480, one implementation of application 480is Affymetrix® LIMS. Full database functionality is intended to providea data streaming solution and a single infrastructure to manageinformation from probe array experiments. Application 480 provides thefunctionality of database storage and retrieval system for accessing andmanipulating all system data. A database server provides an automatedand integrated data management environment for the end user. All processdata, raw data and derived data may be stored as elements of thedatabase, providing an alternative to a file-based storage mechanism. Adatabase back end may also provide integration of application 480 into acustomer's overall information system infrastructure. Data typically isaccessible through standard interfaces and can be tracked, queried,archived, exported, imported and administered.

Application 480 of the illustrated implementation supports processtracking for a generic assay; adds enhanced administration functionalityfor managing synthesized probe arrays, spotted probe arrays, and datafrom these or other types of probe arrays that typically are publishedto a database schema standard such as Affymetrix' AADM standard;provides a full Oracle® database management software or SQL Serversolution; supports publishing of genotype and sequence data; andprovides a high level of security for the LIMS system.

In particular, application 480 of the illustrated example providesprocesses for enabling sample definition, experiment setup,hybridization, scanning, grid alignment, cell intensity analysis, probearray analysis, publishing, and a variety of other functions related toexperimental design and implementation. Application 480 supportsmultiple experiments per sample definition via a re-queuing process,multiple hybridization and scan operations for a single experiment, datare-analysis, and publishing to more than one database. The processdatabase, which may be implemented either as an Oracle or SQL Serverdatabase management system in the illustrated implementation, typicallyis supported by a COM communication layer to the process database. Agene-information database may also be provided to store chromosome andprobe sequence information about the biological item on the probe array,and related information. Another feature, as noted, is publication ofdata in accordance with a database schema that typically is made publicto enable third-party access and software interface development. Forexample, the AADM database schema provides for publication ofAffymetrix® GeneChip® data with support for either an Oracle or SQLserver database management system. Among other structures, tables areprovided in the AADM implementation that provide support for genotypedata.

In particular implementations, a LIMS security database implements arole-based security level that is integrated with Windows NT® userauthentication security. The security database supports role definition,functional access within a role, and assignment of NT groups and usersto those roles. A role is a collection of users who have a common set ofaccess rights to probe array data. In an illustrative implementation,roles may be defined per server/database, and a role member may be amember of multiple roles. The software determines a user's access rightsbased on predetermined rules governing such rights as a function of roleor other variable. A function is a pre-determined action that is commonto all roles. Each role is defined by the functions it can and cannotperform. Functions explicitly describe the type of action that a memberof the role can perform. The functions supported by a newly created roleinclude, but are not limited to, read process data, delete process data,update process data, archive process data, assume ownership of processdata, import process data, export process data, delete AADM data, createa AADM database, and maintaining roles. When a new user is added to arole, they typically have access privileges for their data and read onlyaccess privilege for other user data within the same role. All non-rolemembers are denied all access privileges to role member's data. Whenapplication 480 of the illustrated implementation is installed, at leasttwo roles are created: administration and system user. The installer ofthe system software is added as a user to the administration role and aselected Windows NT® group is added as a user to the system user role.

In accordance with some implementations, a stand-alone application maybe provided to enable user management capabilities. These capabilitiesinclude but are not limited to the following: AADM database creation,publish data deletion, process data deletion, taking ownership ofprocess data, archiving and de-archiving of process data, data export,data import, role management, filter based find, managing expressionanalysis parameter sets, and managing sample and experiment attributiontemplates. Further details are provided in U.S. patent application Ser.No. 09/682,098, incorporated by reference above.

LIMS Objects 490: In the illustrated implementation, LIMS Objects 490 isan object oriented programmers interface into LIMS server application480. In the illustrated embodiment, LIMS objects 490 includes a numberof Application Programmers Interfaces (APIs), generally and collectivelyrepresented as LIMS API's 494, and a number of LIMS servers, generallyand collectively represented as LIMS servers 492. LIMS servers 492 maybe distributed as out of process executables (“exe's”) and LIMS API's494 may be distributed as object type libraries (“tlb's”). Those ofordinary skill in the art will appreciate that various otherdistribution schemes and arrangements are possible in otherimplementations.

LIMS Objects 490 typically may be used by an application developer(represented in FIG. 4 as applications developer 400) who wishes tointegrate in-house or third-party software systems with a LIMS such asLIMS server application 480. For example, it is illustratively assumedthat applications developer 400 works in an enterprise that employs LIMSserver application 480 to manage data related to experiments conductedon probe arrays, which may include any type probe arrays such asGeneChip® probe arrays or spotted arrays (illustratively represented inFIGS. 2 and 4 as probe array 240). It further is assumed forillustrative purposes that LIMS server application 480 is not afull-service system in that it does not provide functions such aslaboratory process scheduling, sample management, instrument control,batch processing, and/or various data mining, processing, orvisualization functions. Alternatively, application 480 may provide someor all of these functions, but applications developer 400 may wish todevelop alternative or supplementary software applications to performall or portions of any of these or other functions, and/or to integratethird-party software applications for these purposes. LIMS objects 490provides developer 400 with tools to customize both the input of datainto, and output of data from, LIMS server application 480.

LIMS objects 490 includes LIMS API's 494. API's 494, in the particularimplementation of LIMS COM API's, includes the following classes:loading list of objects, reading an object, updating/writing an object,deleting an object, processing data, creating AADM-compliant databases,and invoking the analysis controller. API's are also included forobjects, which are used by the previously listed classes.

Some implementations may include, as one of many possible examples ofdata schemes, the AADM database schema. This particular implementationmay be divided for illustrative purposes into four sub-schemas: chipdesign, experiment setup, analysis results, and protocol parameters. Thechip design sub-schema contains the overall chip description includingthe name, number of rows and columns of cells, the number of units, anda description of the units. The experiment setup sub-schema containsinformation on the chip used and the target that was applied. Theanalysis results sub-schema stores the results from expression analyses.The protocol parameters sub-schema contains parameter informationrelating to target preparation, experiment setup, and chip analysis. TheAADM database can be queried for analysis results, protocol parameters,and experiment setup. Similar queries are enabled by Affymetrix® DataMining Tool software, described in U.S. patent application Ser. No.09/683,980, which is hereby incorporated herein by reference in itsentirety for all purposes. The Affymetrix Data Mining Tool also uses asupplementary database called the Data Mining Info database, whichstores user preferences, saved queries, frequently asked queries, andprobe set lists. The Gene Info database, used by Affymetrix MicroarraySuite, stores probe set information such as descriptions of probe sets,sequences that are tiled on an expression array, and user definedannotations. This database also stores lists of external database linksthat allow users to add links to internal/external databases, whichcould be public or private. The SPT, or “spot” file, contains theresults of the image quantification and CSV information integratedtogether.

Computer 350: User computer 350, shown in FIGS. 3, 4, and 5, may be acomputing device specially designed and configured to support andexecute some or all of the functions of scanner control and analysisexecutables 572. Computer 350 also may be any of a variety of types ofgeneral-purpose computers such as a personal computer, network server,workstation, or other computer platform now or later developed. Computer350 typically includes known components such as a processor 555, anoperating system 560, a system memory 570, memory storage devices 581,and input-output controllers 575. It will be understood by those skilledin the relevant art that there are many possible configurations of thecomponents of computer 350 and that some components that may typicallybe included in computer 350 are not shown, such as cache memory, a databackup unit, and many other devices. Processor 555 may be a commerciallyavailable processor such as a Pentium® processor made by IntelCorporation, a SPARC® processor made by Sun Microsystems, or it may beone of other processors that are or will become available. Processor 555executes operating system 560, which may be, for example, aWindows®-type operating system (such as Windows NT® 4.0 with SP6a) fromthe Microsoft Corporation; a Unix® or Linux-type operating systemavailable from many vendors; another or a future operating system; orsome combination thereof. Operating system 560 interfaces with firmwareand hardware in a well-known manner, and facilitates processor 555 incoordinating and executing the functions of various computer programsthat may be written in a variety of programming languages. Operatingsystem 560, typically in cooperation with processor 555, coordinates andexecutes functions of the other components of computer 350. Operatingsystem 560 also provides scheduling, input-output control, file and datamanagement, memory management, and communication control and relatedservices, all in accordance with known techniques.

System memory 570 may be any of a variety of known or future memorystorage devices. Examples include any commonly available random accessmemory (RAM), magnetic medium such as a resident hard disk or tape, anoptical medium such as a read and write compact disc, or other memorystorage device. Memory storage device 581 may be any of a variety ofknown or future devices, including a compact disk drive, a tape drive, aremovable hard disk drive, or a diskette drive. Such types of memorystorage device 581 typically read from, and/or write to, a programstorage medium (not shown) such as, respectively, a compact disk,magnetic tape, removable hard disk, or floppy diskette. Any of theseprogram storage media, or others now in use or that may later bedeveloped, may be considered a computer program product. As will beappreciated, these program storage media typically store a computersoftware program and/or data. Computer software programs, also calledcomputer control logic, typically are stored in system memory 570 and/orthe program storage device used in conjunction with memory storagedevice 581.

In some embodiments, a computer program product is described comprisinga computer usable medium having control logic (computer softwareprogram, including program code) stored therein. The control logic, whenexecuted by processor 555, causes processor 555 to perform functionsdescribed herein. In other embodiments, some functions are implementedprimarily in hardware using, for example, a hardware state machine.Implementation of the hardware state machine so as to perform thefunctions described herein will be apparent to those skilled in therelevant arts.

Input-output controllers 575 could include any of a variety of knowndevices for accepting and processing information from a user, whether ahuman or a machine, whether local or remote. Such devices include, forexample, modem cards, network interface cards, sound cards, or othertypes of controllers for any of a variety of known input devices. Outputcontrollers of input-output controllers 575 could include controllersfor any of a variety of known display devices for presenting informationto a user, whether a human or a machine, whether local or remote. If oneof the display devices provides visual information, this informationtypically may be logically and/or physically organized as an array ofpicture elements, sometimes referred to as pixels. A Graphical userinterface (GUI) controller may comprise any of a variety of known orfuture software programs for providing graphical input and outputinterfaces between computer 350 and a user, and for processing userinputs. In the illustrated embodiment, the functional elements ofcomputer 350 communicate with each other via system bus 590. Some ofthese communications may be accomplished in alternative embodimentsusing network or other types of remote communications.

As will be evident to those skilled in the relevant art, executables572, if implemented in software, may be loaded into system memory 570and/or memory storage device 581 through one of the input devices. Allor portions of executables 572 may also reside in a read-only memory orsimilar device of memory storage device 581, such devices not requiringthat executables 572 first be loaded through the input devices. It willbe understood by those skilled in the relevant art that executables 572,or portions of it, may be loaded by processor 555 in a known manner intosystem memory 570, or cache memory (not shown), or both, as advantageousfor execution.

Scanner Computer 510: Scanner 510 may include elements such as processor655, operating system 660, input-output controllers 575, system memory670, memory storage devices 681, and system bus 690 that may, in someimplementations, have the same characteristics of corresponding elementsin computer 350. Other elements of scanner computer 510 may includescanner firmware executables 672, auto-focus/auto-zero executables 674,calibration executables and data 676, and scanner parameter data 677that will each be described in detail below.

Scanner firmware executables 672 may, in many implementations, beenabled to control all functions of scanner 400 based, at least in part,upon data stored locally in scanner parameter data 677 or remotely inone or more data files from one or more remote sources. For example asillustrated in FIG. 5, the remote data source could include computer 350that includes library files 574, calibration data 576, and experimentdata 577 stored in system memory 570. In the present example, the flowof data to scanner computer 510 may be managed by scanner control andanalysis executables 572 that may be responsive to data requests fromfirmware executables 672.

A possible advantage of including scanner computer 510 in a particularimplementation is that scanner 400 may be network based and/or otherwisearranged so that a user computer, such as computer 350, is not required.Input-output controllers 675 may include what is commonly referred to bythose of ordinary skill in the related art as a TCP/IP networkconnection. The term “TCP/IP” generally refers to a set of protocolsthat enable the connection of a number of different networks into anetwork of networks (i.e. the Internet). Scanner computer 510 may usethe network connection to connect to one or more computers, such ascomputer 350, in place of a traditional configuration that includes a“hardwire” connection between a scanner instrument and a singlecomputer. For example, the network connection of input-outputcontrollers 675 may allow for scanner 400 and one more computers to belocated remotely from one another. Additionally, a plurality of users,each with their own computer, may utilize scanner 400 independently. Itsome implementations it is desirable that only a single computer isallowed to connect to scanner 400 at a time. Alternatively, a singlecomputer may interact with a plurality of scanners. In the presentexample, all calibration and instrument specific information may bestored in one or more locations in scanner computer 510 that may be madeavailable to the one or more computers as they interface with scannercomputer 510.

The network based implementation of scanner 400 described above mayinclude methods that enable scanner 400 to operate unimpaired duringaverse situations that, for instance, may include network disconnects,heavy network loading, electrical interference with the networkconnection, or other types of adverse event. In some implementations,scanner 400 may require a periodic signal from computer 350 to indicatethat the connection is intact. If scanner 400 does not receive thatsignal within an expected period of time, scanner 400 may operate on theassumption that the network connection has been lost and start storingdata that would have been transmitted. When the network connection hasbeen reacquired to scanner 400, all collected data and relatedinformation may be transferred to computer 350 that would have normallybeen transferred if the network connection remained intact. For example,during the occurrence of an adverse situation scanner 400 may lose thenetwork connection to computer 350. The methods enable scanner 400 tooperate normally including the acquisition of image data and otheroperations without interruption. Scanner 400 may store the acquiredimage data of at least one complete scanned image in memory storagedevices 681 to insure that the data is not lost.

In some embodiments, scanner computer 510 may also enable scanner 400 tobe configured as a standalone instrument that does not depend upon acontrolling workstation. Scanner computer 510 may acquire and storeimage data as well as function as a data server to multiple clients forefficient data transfer. For example, scanner 400 may include a harddisk or other type of mass storage medium that may be enabled to holdlarge volumes of image, calibration, and scanner parameter data. Scanner400 may additionally include barcode reader 502 that reads one or morebarcode identifiers from barcode label 1500. Scanner computer 510 mayexecute the scan operations based, at least in part, upon one or moredata files associated with the barcode identifiers, and store theacquired image data on the hard disk. Additionally, scanner 400 mayprovide a network file system or FTP service enabling one or more remotecomputers to query and upload scanned images as well as providing aninterface enabling the computer to query scanner data and statistics.

It will be understood by those of ordinary skill in the related art thatthe operations of scanner computer 510 may be performed by a variety ofother servers or computers, such as for instance computer 350 or LIMSserver 320, or that computer 510 may not necessarily reside in scanner400.

Barcode reader 502: FIG. 5 is a functional block diagram of a simplifiedexample of a scanner-computer system showing scanner 400 under thecontrol of computer 350. An element of scanner 400 may include barcodereader 502 that, among other things, is capable of reading or scanning abarcode that uniquely identifies probe array 240 when it is loaded intoscanner 400 either manually or from auto-loader 443. In someimplementations, barcode reader 502 may be located within scanner 400and disposed so that it reads one or more barcodes associated with probearray 240 when the array is loaded into the scanner. Alternativeembodiments may include an external location of barcode reader 502 thatcould, for instance, include a portable and/or hand-held version that auser could employ to manually scan a variety of barcodes. Each of theone or more barcodes may contain one or more barcode identifiers. Eachof the barcode identifiers read by reader 502 may be used by scannerfirmware 672 and/or by scanner control and analysis executables 572, aparticular implementation of which is illustrated at times herein byreference to Affymetrix® MicroArray Suite software or Affymetrix®GeneChip® Operating System. The barcode identifiers may be used tocorrelate probe array 240 to scanner parameter data 677, experiment data577 created by the experimenter, and/or one or more library files 574 orother data. Library files 574 may, in certain implementations, containinformation including, but not limited to probe array type, lot,expiration date, and scanning parameters that could include the size ofthe probe array, feature size, chrome border dimensions, one or moreexcitation/emission wavelengths, or other data. Library files 574 may bereceived by computer 350 from a variety of sources including one or moreremote sources via network 325 that could include an internet web portalsuch as, for example, the Affymetrix.com web portal provided byAffymetrix, Inc.

A barcode, as is known to those of ordinary skill in the relevant art,represents characters by combinations of bars and spaces and may berepresented in a one or multi dimensional format each having a varietyof possible symbologies. The term “symbology” generally refers to a barcode symbol that includes a pattern of bars and spaces that followspecific standards. A multi-dimensional barcode has the advantage ofbeing able to store a greater capacity of information than aone-dimensional format. For example, one format for use with probearrays may include a widely used two-dimensional barcode format. Thetwo-dimensional barcode format may include a symbology that is wellsupported and may also have built-in error detection. Additionally, atwo-dimensional format may be capable of achieving a sufficientsymbology density to accommodate the amount of information associatedwith a probe array, and furthermore have sufficient built in redundancyto make the probability of a false read negligible. Other type of mediamay also be used to store machine-readable information at a highercapacity than that typically available using one-dimensional barcodes,such as magnetic strips and other magnetic or non-magnetic media knownto those of ordinary skill in the relevant arts. Although the use ofbarcodes is described herein with respect to illustrativeimplementations, it will be understood that various alternative media,symbology, and techniques may be employed in alternativeimplementations.

An illustrative example of a label that contains barcode information ispresented in FIG. 15. Barcode label 1500 may be made of a variety ofmaterials and affixed to the probe array cartridge 720 (illustrated inFIG. 7) by a variety of methods. In the presently described example, thelabel may be constructed of white polyester, and affixed to the probearray cartridge with a permanent acrylic or other appropriate adhesive.Additionally, label 1500 may be constructed so that it demonstrates aresistance to aging, temperature extremes, and moisture to insure thatthe label remain in place and the integrity of the data is maintainedfor extended periods of time as well as to endure the conditions ofprobe array processing and data acquisition.

In one embodiment, barcode label 1500 may be positioned so that it iscentered in a label pocket located on the front face of probe arraycartridge 720. Label 1500 may in one implementation conform to the shapeof the pocket that could, for instance, include a rectangular outerborder with two adjacent corners on a short side cut off symmetricallyat 45 degree angles. Label 1500 may also have one or more areas ofmaterial removed from within the area defined by the outside boundary,such as probe-array cutout 1510, to accommodate associated raised,depressed, or other type of features on probe array cartridge 720. Itwill be appreciated by those of ordinary skill in the art that manydifferent positions and shapes of label 1500 are possible, and that theexamples presented are for illustration only and should not be limitingin any way.

Barcode label 1500 may have a variety of possible features that couldinclude primary barcode 1505, human readable form of primary barcode1507, probe array cutout 1510, and secondary barcode 1520. In oneimplementation, the primary barcode may include information encoded inbarcode symbology such as manufacturer identification, part number,design identification number, expiration date, lot number, and sequencenumber (e.g., a number that is incremented by one each time a label isprinted). Barcode 1505 may also include other information possiblyrelating to experimental data such as experiment number or otherinformation defined by the user for experimental purposes. In someembodiments secondary barcode 1520 may be present and may includeinformation encoded in barcode symbology relating to various parametersfor scanning the probe array, e.g., the size or location of the area tobe scanned, the types of labels that will be emitting radiation from thescanned array, the type or intensity of excitation beam to use inscanning the array, the speed at which to scan or the length of time todwell on the features, and so on. Those of ordinary skill in the relatedart will appreciate that information encoded in barcodes 1507 and 1520are not restricted to the examples presented here and should not belimiting in any way. Probe-array cutout 1510 may represent a part of thelabel that is removed, as described above, so that the probe array isnot obscured by the label. Human readable form of primary barcode 1507may contain one or more data elements from primary barcode 1505 and/orsecondary barcode 1520 so that a user may understand and use the dataelements such as, for instance to manually enter the one or more dataelements. For example, one embodiment may include a raised feature onprobe array cartridge 720 that surrounds the location of the probearray. Probe-array cutout 1510, in this example, may be slightly largerthan the area of the raised feature and of similar shape, so that thelabel may be affixed to the probe array cartridge with the raisedfeature protruding through the label.

Other features that may be included in barcode label 1500 include whatare commonly referred to by those of ordinary skill in the relevant artas quiet zones. The term “quiet zone” generally refers to an area arounda barcode that has the same color and reflectivity as the spaces withinthe barcode. The quiet zone enables the barcode reader to detect theboundaries of the barcode and individual elements of the barcodesymbology. A quiet zone may be any of a variety of widths, and may beconsidered to be a component a barcode symbology. Those of ordinaryskill in the art will appreciate that the features of the quiet zonesneed not be the same for the primary and secondary barcodes.

It will be understood by those of ordinary skill in the relevant artthat many possible methods exist for incorporating barcode informationonto a cartridge or other types of packaging. For example a barcodecould be printed directly onto a cartridge, or alternatively bemanufactured as part of the cartridge itself, or as part of the arrayeither incorporated into probes, into or on the substrate, and so on.Other systems and methods of barcode readers are also possible. One suchexample is described in U.S. patent application Ser. No. 10/063,284,entitled “SYSTEM AND METHOD FOR SCANNING OF BIOLOGICAL MATERIAL USINGBARCODED INFORMATION,” filed Apr. 8, 2002 that is incorporated byreference herein in its entirety for all purposes.

Cartridge Transport frame 505: Another element of scanner 400 includescartridge transport frame 505 that provides all of the degrees offreedom required to manipulate probe array 240 or calibration features442 for the purposes of auto-focus, scanning, and calibration. Those ofordinary skill in the related art will appreciate that the term “degreesof freedom” generally refers to the number of independent parametersrequired to specify the position and orientation of an object.Illustrated in FIG. 7 is an example of probe array cartridge 720 that,in one embodiment, houses probe array 240 or calibration features 442.In one embodiment, transport frame 505 is capable of manipulating thecartridge in four of six possible degrees of freedom (roll 700, pitch703, Z 705 and Y 707). In the illustrated embodiment, it generally isnot necessary to manipulate cartridge 720 for yaw 715 and X 717.

Further examples of cartridge transport frame 505 are illustrated inFIGS. 16-19. FIG. 16 illustrates an example of the entire cartridgetransport frame that is comprised of several elements. The main elementsinclude cartridge holder and pitch and roll mechanisms 1600, Y-stage1623, and Focus stage 1625. Elements of transport frame 505 may beconstructed of a variety of materials that unless otherwise stated may,for instance, include A6061 T6 aluminum alloy or other type of metalwith similar properties. Each of the main elements will be described indetail below.

An example of cartridge holder and pitch and roll mechanisms 1600 isfurther illustrated in FIG. 17. Probe array cartridge 720 is slidablyinserted via cartridge insertion guide 1730 into holder 1600. An opticalswitch may be included that is activated when probe array cartridge 720is inserted into the holder 1600. The optical switch may signal scannerfirmware executables 672 when probe array cartridge 720 has beeninserted. Upon receiving a cartridge present signal, firmwareexecutables 672 may send instructions to engage cartridge clamping balls1725 by actuation of cartridge clamp release lever 1720. Cartridgeclamping balls 1725 may engage conical depressions located on the rearsurface (i.e. side opposite the probe array 240) of probe arraycartridge 720 and may employ leaf springs to apply some degree ofpressure to apply and maintain the integrity of the engagement. The leafsprings may be made from a variety of materials including 400 seriesfull hard stainless steel, or other type of full hardened steel. Thepressure applied by the leaf springs via clamping balls 1725 acts topress cartridge 720 up against pitch finger 1710, fixed finger 1715, androll finger 1750, thereby holding cartridge in place and enablingcontrol of cartridge position as will be described below with respect toroll and pitch. A possible advantage may include making positionaladjustments to bring all of the features of probe array 240 housed inprobe array cartridge 720 into focus by manipulating the position ofprobe array cartridge 720 without changing the positions of otherhardware elements such as the mechanical elements of cartridge holderand pitch and roll mechanisms 1600. After scanning or calibrations havebeen completed, firmware executables 672 may send instructions todisengage cartridge 720 by actuation of lever 1720 to reversibly retractclamping balls 1725 employing a lever cam mechanism driven by one ormore motors, or alternatively by the movement of Y-stage 1623. Oncereleased by clamping balls 1725, cartridge 720 may be removed fromholder 1600 manually or by elements of auto-loader 443 such asreversible rollers 1106 as will be described in detail below.

As described in the example above, three fingers may support andmechanically reference the front surface of (i.e. side where probe array240 or calibration feature 442 may be viewed and/or scanned) probe arraycartridge 720. With a probe array cartridge engaged in holder 1600,fixed finger 1715 may be located at the upper left corner of probe arraycartridge 720, pitch finger 1710 at the lower left corner, and rollfinger 1750 at the upper right corner. Each of fingers 1710, 1715, and1750 may be arranged orthoganally with respect to one another. In oneimplementation, pitch finger 1710 and roll finger 1750 are moveable viatheir associated pitch and roll mechanisms respectively. The pitch androll mechanisms may be implemented as linear step-motor drivenwedge-levers. As illustrated in the example of FIG. 17, the pitchmechanism includes pitch drive motor 1703, pitch wedge 1705, pitch lever1707, and pitch finger 1710, and the roll mechanism is similarlyconstructed with elements 1740, 1745, 1747, and 1750. In the presentlydescribed embodiment, pitch 703 and roll 700 may be adjusted in order tobring all of the features of probe array 240 or calibration features 442into the same plane with respect to the axis of rotation of galvorotation 149, galvo arm 200, and objective lens 145. For example, onepossible implementation of step-motor driven wedge-levers may yield atleast a twelve to one reduction in the magnitude of the input strokefrom motors 1703 or 1740. The reduction in magnitude provides thecapability to make very accurate micro adjustments to the position ofcartridge 720 in the pitch 703 and roll 700 axes respectively. In thepresent example, very small roll 700 and pitch 703 adjustments may benecessary to bring all of the features contained on probe array 240 orcalibrations features 442 into the same plane of focus. Further detailsof roll and pitch adjustment are described below with respect toauto-focus/auto-zero executables 674.

Probe array 240 or calibration features 442 may be brought into bestfocus by adjusting the distance of probe array 240 or calibrationfeatures 442 from objective lens 145. In some implementations, thedistance adjustment may be employed by moving the position of focusstage 1625 in the Z 705 axis. As illustrated in FIG. 19, Z 705 motionmay be achieved, under the control of scanner firmware executables 672,by rotation of precision focus screw 1960 driven by focus drive motor1640. Focus Screw 1960 may be coupled to a solid hinge four bar flexurefocus stage guide 1800 via a free floating nut. In one embodiment, guide1800 is capable of motion transverse to the plane of best focus whilemaintaining the orientation of the probe array with respect to the pitch703 and roll 700 axes. The maintenance of the orientation may be aidedfurther by one or more anti-rotation bar springs 1940 that may apply ameasure of force to guide 1800 if guide 1800 moves in a rotational axiswith respect to the Z 705 axis. For example, the free floating nut mayinclude an anchor bar that is attached to spring 1940. The other side ofspring 1940 may be attached to a fixed stop positioned on guide 1800,where spring 1940 applies pressure against rotational movement of guide1800 but allows movement in a desired axis. Alternatively, themaintenance of the orientation may be aided by a stabilizing anchor armsuch as, for instance, the anchor bar of the floating nut that is causedto engage a fixed pin by spring 1940.

In one possible implementation, focus screw 1960 acts upon Y-stage 1623to push it in a first direction opposed to a force applied by aplurality of flexure guide preload springs 1930. The movement may beaccomplished by focus drive motor 1640 rotating screw 1960 threaded tothe free floating nut. As will be appreciated by those of ordinary skillin the related art, rotating screw 1960 in a first direction results inthe free floating nut moving an a first direction, such as for instancein a direction opposite the force applied by springs 1930. In someimplementations, it may not be desirable that screw 1960 and the freefloating nut interact directly with Y-stage 1623 and/or guide 1800.Various implementations may include an intermediate element such as, forinstance, a ball-shaped element that may have the advantage of allowingfor proper interaction when the components are not positioned at rightangles with respect to one another. The ball-shaped element may beconstructed of hardened steel or other similar material. Y-stage 1623may also be reversibly moved in the opposite direction to the firstdirection by reversing the rotation of focus screw 1960 where themovement of Y-stage 1623 may be aided by the force applied by theplurality of springs 1930. For example, Y-stage 1623 may be attached tothe top of moving load carrier 1820 via bolts, screws, or other methodcommonly used by those in the art, thus load carrier 1820 moves withY-stage 1623 in the Z 705 axis as one unit.

Other elements of an illustrative embodiment of four bar flexure focusstage guide 1800 are illustrated in FIG. 18. Guide 1800 may beconstructed out of a variety of materials including what those ofordinary skill in the related art may refer to as fully annealed steelor fully annealed tool steels. The elements may include one or moregrooves 1805 that are machined into guide 1800. The length and number ofgrooves 1805 defines the spring rate of flexure guide 1805 that may beoptimized to reliably deliver a desired spring rate. Another element mayinclude solid hinge flexure 1830 that hingedly connects moving loadcarrier 1820 with fixed base 1810 to allow for movement in one axiswithout movement in other axes. In one embodiment, flexure 1830 isconstructed of a single piece material using what those of ordinaryskill in the related art refer to as a solid hinge. The solid hinge hasno friction or stiction (a term commonly understood by those of ordinaryskill in the relevant art to refer, generally speaking, to stronginterfacial adhesion present between contacting crystallinemicrostructure surfaces) thus there is no wear or mechanical play in theflexure.

Returning to the illustrative example of FIG. 19, focus travel limitsensors 1920 may be provided to signal scanner firmware executables 672of the relative position of flexure 1800 and/or that a limit of travelhas been reached. Sensors 1920 may include what are commonly referred toby those of ordinary skill in the related art as Hall effect sensors.Hall effect sensors generally refer to magnetic position sensors thatrespond to the presence or the interruption of a magnetic field byproducing either a digital or an analog output proportional to themagnetic field strength. Limit actuator 1910 may include sensor magnet1911 and be attached to guide 1800 by commonly used methods, and thusmoves in the Z 705 axis as guide 1800 and transport frame 505 moves.Sensor magnet 1911 may then be used to reference the position of guide1800 with respect to the position of sensor 1920. Limits of travel maybe further imposed upon flexure 1800 by focus travel safety limit stop1905. Limits to the range of motion allowed for guide 1800 may bedesirable in many implementations because damage to the flexure guide orother components could occur if the focus stage were to be extended toofar. Thus the range of travel allowed may be predetermined, stored inscanner parameter data 677, and defined by the placements of theactuators and stop.

Translation of probe array 240 along the Y-axis may in one embodiment beaccomplished by a precision linear stage, illustrated in FIGS. 16 and 17as Y-stage 1623, coupled to what is referred to as a micro-steppedmotor/driver, open loop drive mechanism or other type of motorizedmechanism, referred to hereafter as Y-stage drive motor 1610. Y-stage1623 may be constructed of a variety of materials including hardenedtool steel, or other types metals designed to resist wear. Additionally,Y-stage 1623 may include a guide element to support and guide cartridgeholder 1600 using various elements such as, for instance, what may bereferred to by those of ordinary skill in the related art as a quad ballbearing track and carriage as well as a recirculating ball screw andnut. Y-stage 1623 may, for example, be positioned so that it rests abovefocus stage 1625, and have cartridge holder and pitch and rollmechanisms positioned above. In the present example, holder 1600 isfixed to Y-stage 1625 so that movement of Y-stage 1625 in the Y-axiswill move the entire holder 1600 assembly, including probe arraycartridge 720. One possible advantage may include independence of themechanisms for position adjustment such that adjustment in one axis isless likely to affect the adjustments in other axes.

Linear encoder scale 1620 may, in some embodiments, be included thatemploys a graduated scale of measurement that is readable by linearencoder sensor 1630. Linear encoder sensor 1630 may be an optical,magnetic, or other type of sensor enabled to read position informationfrom scale 1620. The position information may then be provided tofirmware executables 672 that may be used for accurate control ofY-stage motion reducing the probability of position errors relating tothe Y-axis, as will be described in detail below. For example, duringfluorescence acquisition, the Y-stage may translate probe array 240 ofprobe array cartridge 720 underneath arcuate path 250 of objective lens145. The translation distance for each pass of arcuate path 250 mayinclude 2.5 μm increments that in many implementations of probe array240 enable the complete acquisition of all of the required pixels in theY-axis. In the present example, the Y-stage position informationprovided by sensor 1630 may be used for one or more methods to reduceY-axis position error, including correcting acquired image data byadjusting pixel position placement based, at least in part, uponcalculated error values from measured position information. In thedescribed example, pixel position placement may refer to a computationalmethod for image generation of acquired pixel data, where software, suchas firmware 672, generates an image by placing the acquired pixel datain an appropriate position based on one or more parameters includingy-axis error, to generate an image that accurately reflects the scannedprobe array.

Another implementation of a method for reducing Y-axis position error isillustrated in FIGS. 28 and 29. The implementation includes a method forfirmware 672 control of Y-stage motion using position informationprovided by sensor 1630 that enables very fast interruption of Y-stagedrive motor 1610. As illustrated in FIG. 29, step 2905 enables a Y-Stageincrement mode and identifies a scan line increment. Position determiner2810 receives position increment data 2805, according to step 2905, inorder to calculate the next desired position according to linear encoderscale 1620. Each calculated position corresponds to the line acquisitionpositions that Y-stage 1623 must stop at for the appropriate acquisitionof a line of image data. For example, the Y-stage position incrementscould include 2.5 μm as in a previous example or some other distancerelative to the features of the scanner and probe array 240. In thepresent example, the line acquisition positions according to linearencoder scale 1620 could be 2.5, 5, 7.5 μm, etc. until the entire probearray has been scanned in the Y-axis.

Each calculated position may be received by comparator 2820 asillustrated in step 2910. Comparator 2820 receives a signal to incrementto the next scan line position from scanner optics and detectors 100,illustrated as step 2920, and instructs Y-stage drive motor 1610 to movein the desired direction as illustrated in step 2925. Encoder Sensor1630 reads the position of the Y-stage as illustrated in step 2930, bylinear encoder sensor 1630 and relayed to comparator 2820. When theY-stage 1623 has reached the next calculated position as illustrated instep 2940, comparator 2820 stops the motor increments and holds theY-stage in position for example by using what is known to those in theart as a fixed holding current.

In steps 2945 and 2950, comparator 2820 signals scanner optics anddetectors 100 to acquire a line of scan data and receives a signal whenthe acquisition is complete. Those of ordinary skill in the relevant artwill appreciate that many possible methods of communication betweencomponents are possible and the example is presented for the purposes ofillustration only. As an example, a third component that is independentof the comparator 2820 and scanner optics and detectors 100 may serve asan intermediary for the transmission of signals and commands.Alternatively, comparator 2820 may implement the incremental Y-stagemovement within a desired period of time such as, for instance, 10milliseconds. At the end of the desired period of time, scanner firmware672 may automatically initiate the acquisition of a line of scan data byscanner optics and detectors 100.

As illustrated by decision element 2960, if position determiner 2810signals comparator 2820 to remain in Y-stage increment mode, methodsteps 2910 through 2950 are repeated. Otherwise the method has finishedas illustrated in step 2970, indicating that the probe array has beencompletely scanned.

In some implementations, probe array cartridge 720 generally remains inthe same plane of orientation with respect to scanner 400 from the pointthat it is loaded into scanner 400 to the point at which it is ejected.This may apply to all operations of the scanner including the auto-focusand scan operations. For example, cartridge 720 may be received by thescanner at the load position in a vertical orientation, where probearray 240 would be located on one of the side faces of the cartridge.While remaining in the same vertical orientation the cartridge is placedinto transport frame 505. Probe array 240, housed in probe arraycartridge 720, is positioned into the best plane of focus bymanipulating the probe array cartridge via the pitch 703, roil 700, andZ 705 mechanisms. The probe array is then scanned along Y 707 axis, andthe probe array cartridge is returned upon completion to the loadposition via transport frame 505 in the same vertical orientation thatit was received in.

Auto-Focus/Auto-Zero Executables and Data 674: In the illustratedimplementation, auto-focus/auto-zero executables 674 occur prior toscanning what may be referred to as the active area of probe array 240.The term “active area” as used herein, generally refers to the area ofprobe array 240 that contains experimental probes. There may, in manyimplementations, be a variety of features located outside of the activearea, including chrome border 710, one or more control features, orother elements not directly related to a biological experiment. In theillustrated implementation, auto-focus/auto-zero executables 674 mayexecute a number of operations for the purposes of measuring andadjusting the position of probe array 240 in a plurality of axesincluding pitch 703, roll 700, and Z 705 so that all probe features ofprobe array 240 are in the plane of best focus, and may herein bereferred to as “auto-focuser”. The term “plane of best focus” as usedherein generally refers to an optical plane parallel to the plane ofgalvo rotation 149 where excitation beam 135 is focused to a spot sizethat may correspond to what is commonly referred to as the beam “waist”.As will be described in greater detail below, a spot size in somenon-limiting implementations is a 3.5 μm spot that may be optimal forfluorophore excitation and fluorescent emission detection. It will beunderstood that other spot sizes may be appropriate under theillustrated or alternative implementations and/or experimentalconditions.

Illustrated in FIGS. 7A and 7B is an example of probe array 240 housedin cartridge 720 in which probe array 240 may be bounded by chromeborder 710. In the present example, chrome border 710 may be located onthe inside surface of the glass that covers probe array 240, where boththe chrome border and probe array are in close proximity and are in thesame plane of focus. In one embodiment chrome border 710 may have aminimum width that, for instance, could include a width larger than thediameter of a spot size associated with the focus plane at the maximumrange of travel away from probe array 240 in the Z 705 axis. As those ofordinary skill in the related art will appreciate, a minimum spot sizecorresponds to the beam waist and as the distance increases from thebest plane of focus (i.e. the beam waist), the associated spot sizediameter increases. Thus, the size of a reflected spot may varyaccording to the plane of focus and are generally smallest at the waist,may herein be referred to as “reflection spots”. An embodiment ofauto-focus/auto-zero executables and data 674 may use a measure of thespot diameter to determine the best plane of focus, as will be describedin detail below.

In some implementations of an auto-focus operation, the best plane offocus may be determined, at least in part, using a spot of reflectedlight of predetermined size produced by excitation beam 135 and focusedonto chrome border 710. For example objective lens 145 focusesexcitation beam 135 to a location, illustrated in FIG. 7C as spot 730,and scans along an arc in the direction of the X-axis, illustrated inFIG. 7B as arcuate path 250. Spot 730 is reflected from chrome border710 that in the present example could illustratively be assumed to havea width of 100 μm, although a variety of different widths are possible.In the present example illustrated in FIGS. 7B and 7C, executables anddata 674 may use data corresponding to the intensity of the reflectedlight from spot 730 from a single pass of arcuate path 250 across thetop border of chrome border 710 and subsequently from a single passacross the bottom border. Spot 730 from may cross chrome border 710 atangle θ 1340, where θ 1340 may correspond to an angle between the rangesof 0 and 90 degrees, or 90 to 180 degrees, being more to the extremes of0 and 180 degrees respectively. For example angle θ 1340 may be almosttangential to chrome border 710, creating a large number of data pointsand allowing for calculation of a slope value, as will be discussed indetail below. In the present example, the reflected intensity data usedfor calculating the slope value may correspond to a range of reflectedintensity values starting from the point that spot 730 begins to crosschrome border 710, illustrated as spot 730′, to the point that entirediameter of spot 730 has crossed and is completely reflected by border710, illustrated as 730″.

For the purposes of comparison, those of ordinary skill in the relatedart will appreciate that if, for example, spot 730 crosses the plane ofthe chrome border at an angle of 90 degrees, the duration of timebetween the point when the beam first crosses the plane to the pointwhen the beam has completely crossed plane would be shorter than if theangle was almost tangential to the plane. In the present example, theresult of the shorter time duration would be the collection of a smallernumber of data points and would not allow for calculation of a slopevalue. As will be further described in detail below, having a largenumber of data points may be desirable for a variety of reasonsincluding the reduction of error inherent in the reflected intensitydata.

In one embodiment, the reflected intensity data collected by executables674 may be representative, for instance, of the measured intensity ofreflected spot 730 by detector 192. For example, those of ordinary skillin the related art will appreciate that the reflected intensity may bethe greatest at the beam waist where the light from excitation beam 135is the most concentrated, as will be described in greater detail below.Additionally, the low end of an intensity range from a set of collecteddata points from a single pass of arcuate path 250 over either the topor bottom portion of chrome border 710 may be representative of datacollected when spot 730 partially overlaps the plane of chrome border710 (i.e. spot 730′) and peaks at the high end of the intensity rangewhen the beam has completely crossed the plane of the chrome border 710(i.e. 730″).

An illustrative example of a method that may be used by executables 674is presented in FIG. 7D. The illustrative representation includes a setof collected data points from a single pass of arcuate path 250 overchrome border 710, plotted on a graph and fitted with a line that formsplateau shape 748. The collected data points on plateau 745 correspondto the level of maximum measured intensity as described above. Slope 744of the fitted line may be calculated by executables 674 for the rangebetween the point represented by spot 730′ when the beam first crossesthe plane of chrome border 710 and to the point maximum emissionintensity 745 of plateau 748. Executables 674 may independently performthe method for the top border and the bottom border for a known positionin the Z 705 axis. In the present example, executables 674 stores aplurality of values that may include slope 744 for the top and slope 744for the bottom, and Z 705 position. Those of ordinary skill in therelated art will appreciate that two smaller peaks in the X 717 axis maybe associated with the graph for the collected data from the bottomchrome border due to arcuate path 250. In such a case, the calculationof slope 744 may be made from a single peak chosen either randomly orbased upon one or more characteristics, or alternatively averaged fromboth peaks.

In one embodiment, executables 674 may repeat the above described methodfor a number of successive Z 705 positions. For example, executables 674may perform the first iteration of the method at a distance known to beoutside the plane of best focus that could be useful as a reference.Executables 674 may iteratively perform the method at successiveincremental distances in the Z 705 axis between objective lens 145 andprobe array 240. Executables 674 may then plot calculated slope 744 foreach Z 705 position on a new graph independently for the collected datafrom the top and bottom, as is illustrated in FIG. 7E. In the presentexample, it may be desirable for the purpose of reducing error toeliminate data points from the high and low extremes of the slope valuesand fit a line to the remaining data points. The data points used forcalculations could, in one possible implementation, be located in range750 that may, for instance, include a range between sixty and seventypercent of the maximum value. Executables 674 may then calculate and fitparabolic line 770 to the data points within range 750. In this example,the plane of best focus corresponds to the Z-axis position associatedwith the maximum height 760 of parabolic line 770 that represents thedistance when the minimum beam spot size is achieved corresponding tothe highest measured intensity. Alternatively, in some implementationsthe height at which the laser spot size is the smallest may not be theheight at which the amount of reflected laser light collected is amaximum due to characteristics of one or more of the optical elements.Instead one or more of the pinholes may be positioned so that the focusheight is one for which “best compromise” of light collection, lightfiltering, and PMT response for the range of wavelengths emitted by aparticular fluorophore is achieved.

In some embodiments it may, for example, be desirable to first performthe auto-focus methods described above to bring a first chrome borderthat could be either the top or bottom chrome border, into the bestplane of focus. Secondly, perform the methods to bring the second chromeborder not adjusted in the first iteration of the method into the bestplane of focus. Then, the auto-focus methods may be repeated upon thefirst chrome border to compensate for error introduced while adjustingthe second border. Additionally, it may be desirable to use convolutionmethods that may, for instance, be used to compare the shape of line 770to an expected shape associated with a desired result. The term“convolution” as used herein, generally refers to one or more methodsthat include an integral which expresses the amount of overlap of onefunction g as it is shifted over another function θ, and therefore“blends” one function with another.

In many implementations, objective lens 145 forms what is referred to asa convergent/divergent beam that, for example, may resemble two coneshapes joined together at their points. The beam's waist (the locationwhere the points of the cones join) correspond to the minimum spot sizedefined in the illustrated implementation as the plane of best focus.Executables 674 may adjust the Z 705, pitch 703 and roll 700 positionsof the array individually or in a variety of combinations using thecorresponding elements of transport frame 505 to achieve best focus. Asprobe array 240 is moved closer to best focus, the laser spot sizeapproaches the minimum spot size for both the top and bottom borders.Once auto-focus executables 674 determines that the array is in bestfocus, no further motion is applied in Z 705, pitch 703 or roll 700. If,for example, after the completion of the auto focus operationmodifications are made to the position of probe array cartridge 720,then executables 674 will perform the auto-focus operation again toposition probe array 240 in the best plane of focus. In someembodiments, dynamic focusing of the array typically need not occurduring scanning, as the plane of focus is not affected as cartridge 720is translated along the Y 707 axis.

Auto-focus/auto-zero executables 674 may require number of datavariables that for instance may be stored locally in scanner parameterdata 677 to perform its operations. One or more elements of scannerparameter data may have been imported from a remote source such as fromlibrary files 574; intermediate results, lab data, and image data 401A;or other remote source of data. An example of one of the many possibledata variables passed into executables 674 may be probe array type datathat may define the physical parameters that executables 674 uses toaccurately position the probe array in the best plane of focus. In thepresent example the probe array type data variable may be provided toexecutables 674 by barcode reader 502, or may alternatively be stored inone or more data files that, for instance, may have been created byexperimenter 475 and identified by a barcode identifier. The datavariables may be categorized by the routines they perform that couldinclude, but are not limited to, Y 707 position of the top edge of thebottom chrome, Y 707 position of the bottom edge of the top chrome,nominal thickness of chrome border 710, distance from the inside edge ofchrome border 710 to the edge of the active area, width of the scan,laser power, PMT gain, Z 705 step size, and Z 705 position used forsearching for chrome. Those of ordinary skill in the relevant art willappreciate that variables may be involved in one or more categories, andthat a variables use in one category may not limit its use in anothercategory.

In some alternative implementations, variables and other aspects of theauto-focus operations may be stored and executed by scanner control andanalysis executables 572, stored in system memory 570 of computer 350and communicated to scanner 400 through input-output controllers 575.Variables and other related data may in some implementations also bestored in experiment data 577, or other memory storage located oncomputer 350 or remotely from it. For example, some auto-focusoperations could include instructions that may be based in part on theprobe array type, and sent to transport frame 505 for the purposes ofmaneuvering the probe array into the appropriate position. In thepresent example other auto-focus operations could also include theinitiation and control of the movement of the scanning arm, laseractivation and collection emitted light, calculation of emitted lightintensities and the plane of best focus. The previous examples arepresented for illustration only, and are not intended to be limiting inany way.

In one embodiment, executables 674 may additionally perform auto-zerooperations prior to an active area scan of each probe array to maximizewhat is commonly referred to by those of ordinary skill in the relatedart as the signal to noise ratio. In other words enhance the differencebetween noise in the system and signal from detected emissions. Theremay be many sources of noise that could affect the resolution of anacquired signal (i.e. the noise may mask all or part of a signal)including local radio or television stations, equipment plugged into thesame power source, nearby conductors carrying “dirty” signals, darkcurrent of components within scanner 400 such as PMT detectors, or othersources of noise. In some implementations, the auto-zero operations maybe carried out subsequent to the auto-focus operations or vice versa.

In the presently described embodiment, scanner 400 may have a largedynamic range capable of detecting very small and very large signals(i.e. detection of low and high levels of fluorescent emission). Forexample, scanner computer 510 may include what is referred to by thoseof ordinary skill in the related art as a 16-bit data acquisition systemthat allows for detection of over 65,000 different levels offluorescence detection. Variations in signal output from noise may bemeasured and can range from 4-8 bits, commonly referred to as leastsignificant bits (LSB) as opposed to the bits that are closer to 65,000,commonly referred to as Most Significant Bits. In the present example,adjusting what those of ordinary skill in the art commonly refer to aschannel offset value of detectors 110, 115, and 180, may be made tomodulate the output from a detector to compensate for such variations.Alternatively, in some implementations it may be desirable to adjust thechannel offset value of laser 120. Those of ordinary skill in therelated art will appreciate that the term “channel offset” generallyrefers to the constant frequency difference between a channel frequencyand a reference frequency. In some implementations it is desirable thatthe channel offsets be adjusted so that the noise is close to, butmeasurably above, zero LSB because values below zero LSB cannot bemeasured.

For example, it can be assumed for illustrative purposes that thevariation present at a given point in time follows a normal or gaussiandistribution. Thus, most of the variation values are clustered close toan average value of all of the variation present. If plotted on a graph,the result would look like a bell shaped curve with the average value ofthe variation being the peak of the curve. In the present example, onestandard deviation of a gaussian distribution may be equal to 32% of thepixels being below zero LSB. Three standard deviations from the averagemay results in less than about 0.3% of the pixels being below zero LSBand satisfies the desired embodiment of having most of the pixelvariation close to zero but measurably above. Those of ordinary skill inthe related art that the term “standard deviation” commonly refers to astatistic that tells you how tightly all the various examples areclustered around the mean in a set of data.

In one embodiment of the auto-zero operation performed by executables674, shutter 134 is closed and in some cases filter wheel 160 is turnedto a position where all light to detector 115 is blocked. Signal 192 isthen representative of the variation or noise present in the system.Executables 674 then makes the calculations based, at least in part,upon that variation and adjusts signal 192 accordingly. For example,after all possible sources of light are blocked, the channel offset ofdetector 115 could be set to a position where the variation istheoretically calculated to be above 0 LSB. The detector could take theequivalent of 60,000 readings of fluorescence and the average valuecould then be calculated. If that value is less than or equal to zero,then the offset value is increased by a predetermined increment such as,for instance, one or more standard deviation values, and new readingsare taken using the method described above. Otherwise the average valueis above zero and the offset value may be further adjusted based upon avalue calculated by executables 674 that may include the differencebetween the measured average and a value that is three times thecalculated standard deviation value for the measured variation.Executables 674 may repeat the method and perform adjustments to thechannel offset value until the channel offset settings have beenreached. Additional examples of noise calculation and compensation maybe found in U.S. Pat. No. 6,490,578; and in U.S. patent application Ser.No. 10/304,092, titled “System, Method, and Product for Dynamic NoiseReduction in Scanning of Biological Materials”, filed Nov. 25, 2002,each of which is hereby incorporated by reference herein in it'sentirety for all purposes.

Auto-Loader 443: FIGS. 8-14 provide an illustrative example of onepossible embodiment of an automatic cartridge loader used in conjunctionwith some implementations of scanner 400. Auto-loader 443 may include anumber of components such as cartridge magazine 1000, cartridgetransport assembly 1105, cooled storage chamber 960, and warm thermalchamber 940. A implementation may include the construction ofauto-loader 443 as a two piece structure that has upper and lowersections.

The upper structure may contain components including heat exchanger 925;fan 926; warm thermal chamber 940 including thermally insulatedpartition 927, and chamber heating element 950; cartridge magazine 1000including hub 1003, compartments 1010, partitions 1002, andcircumferential band 1004; and insulated cover 828 that encloses cooledstorage chamber 960. The lower structure could contain components suchas cartridge transport assembly 1105 including reversible rollers 1106,actuation components 1110, and 1113, and spur gears 1107; andelectronics. It will be understood that various configurations anddistributions of these and other elements are possible, and that someelements may be combined or split into separate elements in alternativeimplementations.

Auto-loader 443 may be covered by insulated cover 828, that for examplecould include elements such as a door with a solenoid actuated latch andvibration dampened hinge to allow access for a user to load or unloadindividual cartridges as well as magazine 1000. In some implementationsthe solenoid actuated latch may be responsive to instructions fromfirmware executables 672. For example, firmware executables 672 mayinstruct the solenoid actuated latch to allow access at any time whenscanner 400 is not performing any scanning function or operation.Alternatively, the solenoid actuated latch may be responsive toinstructions from computer 350 via firmware 672 that may be initiated byexperimenter 475. In the present example, experimenter 475 may wish tointerrupt the operations of scanner 400 for a variety of reasons suchas, for instance, to add or remove one or more cartridges 720 ormagazine 1000, or to introduce a probe array cartridge directly intoscanner 400 for immediate scanning. Experimenter 475 may use computer350 to instruct firmware executables to interrupt one or more operationsand release the solenoid actuated latch. After experimenter 475 hascompleted the desired task, user 475 may make a selection via computer350 enabling firmware executables 672 to resume the one or moreoperations.

Some features of the embodiment include the preservation of biologicalintegrity of the probe arrays for a relatively long period of time, suchas sixteen hours or more, by controlling one or more environmentalparameters of the array storage environment. Also, in the event of apower failure or error condition that prevents normal operation ofscanner 400, auto-loader 443 may indicate the failure to the user andmaintain a storage temperature for all probe arrays within cooledstorage chamber 960 and/or warm thermal chamber 940 using anuninterruptible power supply system. Other features of the embodimentmay include pre-heating probe array cartridge 720 to the sametemperature as the internal environment of scanner 400 prior totransport into scanner 400.

The features described above provide optional equipment and techniquesto transfer a plurality of probe array cartridges 720 from a temperaturecontrolled storage environment into scanner 400 in an organized andefficient manner, and to return the cartridges to the temperaturecontrolled storage environment following imaging. Optimal temperaturesfor storing cartridges may vary, but typically include temperaturespreferably ranging from 2° C. to 15° C. FIG. 9 provides an illustrativeexample of auto-loader 443 positioned above scanner 400 secured by aplurality of mounting bosses that may be inserted through the scannercover and fastened with self-retaining, floating hardware. In oneembodiment, auto-loader 443 may be enabled to receive its electricalpower from scanner 400. One possible method of electrical connectionbetween scanner 400 and auto-loader 443 may include what is commonlyreferred to as a blind-mate connector that includes shielding that couldbe pass through a specialized opening in the scanner cover and connectedto an internal power source within scanner 400. Alternative embodimentsof auto-loader 443 could include an independent power supply dedicatedto auto-loader 443 or other power supply method.

The embodiment may also include heat exchanger 925, enabled to providecooling for cooled storage chamber 960. In many implementations it maybe desirable to maintain a temperature range such as, for instance, arange between 2° C. to 15° C. to maintain the biological integrity ofhybridized probe arrays. Fan 926 may be enabled to circulate the cooledair from heat exchanger 925 around one or more probe array cartridges720 positioned in magazine 1000. In one embodiment of the invention, oneor more thermostats may be included to provide temperature measurementsto firmware executables 672 of the air surrounding the probe arraycartridges 720 and/or the ambient air temperature associated with theinternal environment of scanner 400. Firmware executables 672 mayactivate exchanger 925 as necessary to maintain a desired temperature,and additionally provide sufficient cooling so that probe arraycartridge 720 could be cooled to the desired temperature in one hour orless. An alternative embodiment may include a more direct means oftemperature measurement such as, for instance, measuring the temperatureof probe array cartridge 720 using an infrared detector. An infrareddetector, as it is known to those of ordinary skill in the relevant art,is enabled to detect temperature from a specific location and be tunedto a specific temperature range so that thermal noise from air or othersources will not affect it. For example, an infrared temperaturedetector may not require physical contact with a probe array cartridgeto specifically read its temperature. The infrared detector may be aimedat a specific location on a probe array to detect the temperature atthat location. In the present example, the infrared detector detects theinfrared energy (i.e. heat energy) emitted by the probe array cartridge.

Also illustrated in FIG. 9 are thermally insulated partitions 927 thatmay be enabled to separate warm thermal chamber 940 from cooled storagechamber 960. In one implementation, thermally insulated partitions 927are aligned radially with cartridge magazine hub 1003, and aligned withpartitions 1002. When properly positioned a seal is formed betweenpartitions 927, partitions 1002, and insulated cover 828, thus thermallyisolating warm thermal chamber 940 and a plurality of compartments 1010from the remainder of magazine 1000. Firmware executables 672 mayprovide temperature control to warm thermal chamber 940 using chamberheating element 950. For example heating element 950 may include arecirculating fan and what may be referred to as a heat sink orresistive heater to warm the air. In the present example, chamber 940may be enabled to warm a plurality of probe array cartridges quickly sothat, for instance, within a time period of ten minutes or less the oneor more probe array cartridges within chamber 940 may be warmed to thetemperature.

In the presently described embodiment, previously cooled air from cooledstorage chamber 960 may be used by chamber heating element 950. Thepreviously cooled air may enable warm thermal chamber 940 to preventcondensation from forming on the surface of probe array cartridge 720.It will be appreciated by those of ordinary skill in the related artthat warm air is capable of holding more water vapor than cold air, thuscooling often forces the air to lose some measure of water vapor (i.e.become drier) depending upon how cold the air becomes. It may bedesirable in many implementations to prevent condensation formation fora variety of reasons including the distortion of images and/or theobstruction of probe array features from the view of scanner optics anddetectors 100.

Warm thermal chamber may, in one embodiment, be enabled to hold aplurality of probe array cartridges 720. Holding a plurality ofcartridges may provide sufficient time for the temperature of a probearray cartridge to change from the temperature of cooled storage chamber960 to the temperature of warm thermal chamber 940 prior to being loadedinto scanner 400. For example, warm thermal chamber 940 may have thecapacity to hold two probe array cartridges. In the present example afirst cartridge may be positioned over the load point and loaded intoscanner 400 by transport assembly 1105, while a second cartridge 720remains in a waiting position in warm thermal chamber 940. The firstcartridge may be loaded into scanner 400 via transport assembly 1105,and subsequently returned after the completion of scanning operations.Firmware executables 672 may then advance magazine 1000 by one positionof compartment 1010 so that the first cartridge is now moved out of warmthermal chamber 940 and into cooled storage chamber 960. The secondcartridge that was in the waiting position in chamber 940 has beenwarmed to the temperature of warm thermal chamber 940 and moved to theloading position. A new, third cartridge has entered warm chamber 940into the waiting position from cooled storage chamber 960 and begins towarm. The previous example is for the purposes of illustration only andit will be appreciated to those of ordinary skill in the art that manyvariations may exist.

Warm thermal chamber may use similar methods of temperature measurementas those described above with respect to cooled storage chamber 960.Returning to the previous example of using an infrared detector formeasuring the temperature of a probe array cartridge, the temperature ofthe probe array cartridge is precisely measured and the informationimmediately relayed to firmware executables 672. This enables firmwareexecutables 672 to respond to minute temperature variations that mayexceed ideal limits. Thus the potential of overheating a probe arraycartridge may be reduced and higher air temperatures could be used toheat the probe array cartridge more quickly. In the present example, theinfrared detector could be aimed at a plurality of possible locations onprobe array cartridge 720 including the glass over the probe array, thetop or bottom of the outer exposed edge of the probe array cartridgewhen loaded into the magazine 1000, the bottom edge of the probe arraycartridge that could be viewed through an additional opening in base1314, or other location on the probe array cartridge.

In one embodiment, firmware executables 672 may instruct auto-loader 443to alter its mode of operations between a plurality of different modes,where each mode may have distinct utility and operating parameters. Forexample, auto-loader 443 may change from a scanning mode to a long-termstorage mode after all probe array cartridges contained in magazine 1000have been scanned. In the present example, when auto-loader 443 entersthe long term storage mode, heating element 1550 is turned off andcartridge magazine 1000 periodically advances by one or more positionsof compartments 1010 to bring all the probe arrays contained in magazine1000 to an equal and cool temperature. The capability to change modesenables the user to set up one or more experiments and leave it for anextended period without compromising the data collection process orintegrity of the biological samples. The user may use computer 350 toset up and store one or more experimental parameters in experiment data577 that scanner control and analysis executables 572 may then provideto firmware executables 672.

FIG. 10 illustrates an example of cartridge magazine 1000 that in oneembodiment is removable from auto-loader 443. Magazine 1000 may becapable of holding a range of one or more cartridges, for instance, arange from 1 to 100 cartridges is possible. Although it is commonlyunderstood that many other possible ranges exist. For example, someembodiments could include a cartridge magazine that holds up to 32 or 48cartridges. Additional components and features of magazine 1000 mayinclude partitions 1002 that hold and maintain separation between probearray cartridges 720. It may be desirable that magazine include abarcode with an associated barcode identifier that could, for instance,be used to identify one or more of the probe array cartridges containedin magazine 1000, identify experiments, or other type of information. Inone embodiment each of compartments 1010 may hold a single probe arraycartridge, although alternative implementations may include each ofcompartments 1010 holding 2, 3, or more probe array cartridges. Theseparation provided by partitions 1002 includes physical and thermalseparation, where partition 1002 may act as a thermal barrier betweenthe compartments. Another component includes hub 1603 in the center ofmagazine 1600 that is the center of rotation of the cartridges about thevertical axis of the magazine. Also, circumferential band 1604 encirclescartridge partitions 1602 providing structural support for magazine1600.

Probe array cartridges 720 may be oriented vertically or horizontally inmagazine 1000, and loaded with no sequential relationship to oneanother. For example, scanner 400 may include a barcode reader that iscapable of reading one or more barcode identifiers from barcode label1500 that firmware executables may use to identify the specific probearray cartridge 720. Also in the present example, it may not be requiredthat each of compartments 1010 contain a probe array cartridge.Auto-loader 443 may include one or more sensors such as, for instance,an optical sensor that may enable firmware executables 672 to recognizeand skip empty locations in magazine 1000.

In some embodiments, cartridge magazine 1000 may implement a variety ofdifferent methods for the orientation and movement of probe arraycartridges 720. For example, probe array cartridges 720 may be orientedhorizontally in magazine 1000 on one of the side edges of the cartridge.In a separate example, the cartridges may be in a different style ofmagazine where the cartridges could be positioned so that they lie flaton the front or back face of the cartridge. The magazine could beconstructed such that the cartridges may be stacked verticallyresembling a deck of cards. In the present example a cartridge from thebottom of the stack could be removed for scanning operations, advancingthe remaining cartridges in the stack down by one position. The removedcartridge could then be placed back on the top of the stack, oralternatively be added to a different stack.

Cartridge magazine 1000, including partitions 1002, hub 1003, and band1004, may be constructed of plastics, metals, or combinations thereof,or other appropriate materials. For example one embodiment could includecartridge magazine 1000, including the hub 1003, partitions 1002, andcircumferential band 1004, comprised as a single piece ofinjection-molded plastic such as ABS.

FIG. 11 is an illustration of cartridge 720 located within cartridgetransport assembly 1105. Cartridge transport assembly 1105 includesreversible rollers 1106 that manipulate cartridge 720. Spur gears 1107are driven by a motor (not shown) to power the motion of rollers 1106. Ahousing comprising a front plate 1108, side plates 1110, and rear plate1111, as well as actuation components 1113 that activates the captureand release of cartridge 720 by rollers 1106. Similar to magazine 1000,cartridge transport assembly 1105 may be constructed of a variety ofmaterials including plastics, metals, or combinations thereof, or otherappropriate materials. For example, plates 1108, 1110, and 1111 as wellas parts of rollers 1106 and actuation components may be constructed ofaluminum or other type of metal while elements such as spur gears 1107and/or parts of rollers 1106 may be constructed of plastic.

Illustrated further in FIG. 12 is an example of how cartridge 720 androllers 1106 may interact. In the illustrated example rollers 1106 maybe extended towards and engage one or more fiducial features of probearray cartridge 720 via actuation components 1113. Movement of therollers in a first direction will impel the cartridge downwards, whilemovement of the rollers in the reverse direction will impel thecartridge upwards. Rollers 1106 may be disengaged and retracted fromprobe array cartridge 720 via actuation components 1113. In oneembodiment, reversible rollers 1106 may reversibly translate probe arraycartridge 720 across the distance between magazine 1100 and scanner 400.

FIGS. 13 and 14 further provide an illustrative example of theinteraction between cartridge transport assembly 1105, cartridgemagazine base 1314, and cartridge magazine 1000. Cartridge magazine base1314 includes slot 1315 that is associated with the loading position andmay provide for through-passage of a selected probe array cartridgebetween magazine 1000 and cartridge holder 1600 of scanner 400.Cartridge magazine base 1314, in conjunction with partitions 1002 areenabled to support and hold the one or more probe array cartridges inplace. When probe array cartridge 720 is positioned in the magazineloading position over slot 1315, it may be supported on the underside bysliding door 1316. Under the control of firmware executables 672,sliding door 1316 may block or allow passage of the probe arraycartridge through transport assembly 1105. For example, firmwareexecutables may extend reversible rollers 1106 and engage probe arraycartridge 720. In the present example rollers 1106 may be enabled toreach above the level of cartridge magazine base 1914 with sliding door1316 in the closed position and engage the one or more fiducial featuresof probe array cartridge 720. Firmware executables 672 may then instructlinear motor 1423 to actuate sliding door 1316, opening it and enablingrollers 1106 to positionally control the probe array cartridge. Firmwareexecutables 672 then instructs rollers 1106 to impel the probe arraycartridge through transport assembly 1105 to scanner 400. Once probearray cartridge 720 is properly positioned in scanner 400 and/orreceived by cartridge holder 1600, firmware executables 672 instructsrollers 1106 to disengage from the probe array cartridge and retract.Rollers 1106 may also be enabled to accept the probe array cartridgefrom scanner 400 in a similar manner to that described above, and returnit to the same or different compartment 1010 of magazine 1000 asdirected by firmware executables 672.

Further illustrated in FIGS. 13 and 14 is hub base 1317, positionedbeneath hub 1003 that may further be attached to a shaft connected to atiming belt pulley (not shown). The timing belt pulley may be driven viaa timing belt by a motor such as linear motor 1423 or some other motor,for the purposes of advancing magazine 1000. Additionally, pin 1318 maybe provided to maintain the positional orientation of cartridge magazinebase 1314 such that slot 1315, and hence the loading position, does notmove when cartridge magazine 1000 is advanced.

For example, cartridge magazine 1000 advances a first selected cartridgeinto the loading position above slot 1315 by rotating cartridge magazine1000 about its vertical axis. A sensor may be included such as, forinstance, an optical or other type of sensor that establishes that aprobe array cartridge is properly aligned in the loading position. Thecartridge may be lowered through cartridge transport assembly 1105 andinto scanner 400 by means of rollers 1106. After the operations ofscanner 400 have been completed, the probe array cartridge is reversiblytransported from the scanner to the loading position in magazine 1600.The magazine advances, moving the first probe array cartridge from theloading position and moving a second probe array cartridge into theloading position where the probe array cartridge is positioned aboveslot 1315 and rollers 1106.

Those of ordinary skill in the relevant art will appreciate that manypossible methods and components exist for the storage and automatictransport of probe array cartridges. Two examples are described in U.S.Pat. No. 6,511,277 titled “CARTRIDGE LOADER AND METHODS”; and U.S.patent application Ser. No. 10/180,588, titled “Cartridge Loader andMethods”, filed Jun. 26, 2002, both of which are hereby incorporatedherein by reference in their entireties for all purposes.

In some embodiments it may be desirable and/or required that scanner 400be operated without an implementation of auto-loader 443. One embodimentof scanner 400 includes elements that allow for a user to manuallyload/unload one or more probe array cartridges 720. The includedelements also work in conjunction with an implementation of auto-loader443. For example, the elements may include a passive door mechanism thatdoes not use a motor to open or close the door. Instead, a user or animplementation of auto-loader 443 may manually actuate the door.Additionally, the door may also be recessed into the housing of thescanner limiting the possibility of opening at inappropriate times.

Calibration of Scanner: It is desirable in many embodiments to calibratethe scanner to compensate for variations in components that could affectthe proper acquisition of image data. Such variations could includethose caused by mechanical wear or changes to one or more opticalelements. Those of ordinary skill in the related art will appreciatethat many optical elements may be affected by a variety of factorsincluding temperature fluctuation, mechanical wear, componentimperfections, slight changes caused by physical insult, or other of avariety of possible factors. In one implementation, scanner 400 could beinitially calibrated at the factory then again during routine service inthe field. Alternatively or in addition to the previous implementation,the calibration operation could be automatically performed prior to orduring each scan, during the power-up routine of the scanner, or atother frequent intervals. In one embodiment, scanner 400 is calibratedfor the spatial characteristics of mechanical movement as well asoptical characteristics that include calibration of light collectioncomponents and radius of the light path calculations. Additionally, inthe same or other embodiment, scanner 400 could be calibrated frequentlyto provide the most accurate images where the calibration procedureswould not require user initiation or intervention.

Examples of some possible compensation mechanisms for image intensitycalibration could include gain adjustments of the photo multiplier tube,power adjustments to the laser, or other adjustments to the optics anddetectors. Similarly mechanical compensation to reduce error to lessthan one pixel could include adjustments to the speed of the scanningarm at specific points in the arc, adjustments to the movement oftransport frame 505, calculated corrections to the acquired image databy scanner firmware executables 672 or scanner control and analysisexecutables 572 such as the implementations, Affymetrix® MicroarraySuite and Affymetrix® GeneChip® Operating System.

In one possible implementation, calibration features 442 may be used tocalibrate scanner 400. For example, calibration features 442 could behoused in a cartridge and inserted into scanner 400 in a similar mannerto probe array cartridge 720. Calibration features 442 could include aplurality of calibration arrays or configurations that may each includevarious geometric chrome, fluorescent, or other type of featuresdeposited on the inside surface of the glass and/or on the substrate ofprobe array 240. For example, one embodiment may include the placementof calibration features 442 such that they are in the same plane asprobe array 240. In the present example, calibration features 442 mayinclude a pattern of chrome features where a fluorescing referencemedium may be deposited on top of the pattern of chrome features andloaded into scanner 400. Scanner 400 performs a calibration operation byscanning at the excitation wavelength of the fluorescent referencemedium through the glass cover. One or more detectors of scanner 400receives the emitted light from the reference medium, where thereflected light from the chrome features masks the fluorescent emissionsof the reference medium. Thus the chrome pattern may be used as anegative image for calibrating scanner 400. Scanner 400 mayalternatively image the calibration features by detecting the reflectedlight from the chrome features at the excitation wavelength that allowsthe chrome pattern to be used as a positive image. Additionally, asscanner 400 performs the described calibration operation, both intensitydata and position data are recorded as digital data in memory. Theintensity data could be based on fluorescent emissions and/or diffusereflected light and the position data may be include image data of thechrome features, data from galvo transducer 515, and/or the knownposition data of the chrome features that may for instance be stored incalibration data 576.

Those of ordinary skill in the relevant art will appreciate that thegeometric features of calibration features 442 may be constructed in avariety of different configurations for a variety of calibrationmethods. For example, one possible implementation for what may bereferred to as horizontal linearity calibration may include calibrationfeatures 442 being disposed on a substrate in a grating or ladderpattern. The ladder pattern may be constructed of chrome elements ofknown characteristics that may resemble a series of vertical barsarranged in a horizontal pattern with each bar spaced an equal distanceapart. In the present example, it may be desirable that the position ofthe leading edge and width of each of the bars is exactly known.Alternatively a similar configuration may used for what is referred toas vertical linearity calibration that includes the grating or ladderpattern of bars oriented in a vertical pattern instead of or in additionto the horizontal pattern. Those of ordinary skill in the related artwill appreciate that the term “linearity calibration” generally refersto methods for the correction of error in one or more linear axes. Forinstance, horizontal linearity correction methods may include correctingfor X-axis pixel placement error in an acquired image.

Continuing the example described above, the one or more linearitycalibration arrays may be scanned and an image of pattern acquired alongwith positional data from galvo position transducer 515. Calibrationexecutables and data 676 may then compare the image positions ofcalibration features 442, galvo position, and the known positions ofleading edge of calibration features 442, and generate an error tablethat include one or more image corrections values for each pixelposition of an acquired image. Calibration executables and data 676 maycorrect the placement of each pixel in an acquired image of probe array240 based, at least in part, upon the error table that may, forinstance, specify that a particular pixel be “moved” (e.g., associatedin a database or other data storage format with a different, often adisplaced, position on the array) by whole or fractional pixelincrements in the plus or minus direction in the X and or Y axes.Methods of linearity correction are described in greater detail below inreference to the methods illustrated in FIGS. 20 through 22.

Also, possible implementations may include calibration features 442 asincorporated elements of probe array 240. For example, some embodimentsof probe array 240 may include one or more features used for the purposeof determining the efficiency of hybridization of the biological sampleswith specific probes. The specific probes may detect a target moleculethat is added during the experimental protocol, or alternatively maydetect an mRNA signal from one or more genes known to be expressed athigh levels such as, for instance, a gene important for cell structurethat could include β-actin. In the present example, the image quality ofthe one or more features may be associated with the hybridizationconditions present during an experiment. Additionally, the one or morefeatures could be used as anchor positions for methods of geometriccalibration and/or methods of collected emission intensity calibrationof one or more elements of optics and detectors 100 using the collectedlight emitted from the hybridized probes.

In an alternative example, the biological probes in the active area ofprobe array 240 may serve to calibrate scanner 400. One possibleadvantage includes calibrating scanner 400 by using the same fluorescenttags as used to collect experimental data. Thus, by utilizing the samefluorescent tags the accuracy of the calibration method is improved byeliminating the variation caused by the characteristic emission spectraof different fluorophores. A table for geometric calibration could beconstructed by finding the center pixel location of the probe arrayfeatures by known methods and plotting on a graph against the knownlocation of that center pixel of the probe feature on the X and Y axes.The differences between the measured and known expected locations arecalculated as error correction values and entered on a table that mayfor instance be stored in calibration executables 676. Intensitycalibrations could be made in the present example by using the ratio ofmeasured emissions from what may be referred to as the match andmismatch probes.

Additionally, calibration features 442 could include one or morecalibration arrays used to validate the effectiveness of both thehorizontal and vertical linearity calibration methods. For example, theone or more calibration arrays may include an array that provides apattern that perfectly corresponds to the placement of a grid patternsuch as, for instance, a grid pattern that similarly corresponds to thebiological probe features of probe array 240. After the pattern isimaged by scanner 400 and image correction methods are applied, the gridpattern may be placed on the image. A user may then visually see if thegrid is properly aligned to the image, and if, for instance, it is notproperly aligned then scanner 400 may be recalibrated for horizontaland/or vertical linearity.

Calibration features 442 may also include one or more calibration arraysfor calibration of mechanical or other type or error with respect toroll 700 and pitch 703. For example, roll 700 and pitch 703 calibrationmay be performed by a variety of methods. One such method includes usinga calibration array disposed in a housing similar to probe arraycartridge 720 that is either known to be perfectly flat or that the rolland pitch error associated with the test chip has been preciselymeasured. Methods of measurement may include using fiducial features ofprobe array 240 such as, for instance, chrome border 710 or placingadditional reference targets in specific positions on or around probearray 240. The specific positions could include the corners of probearray 240 where the roll and pitch of the entire probe array may bemeasured in addition to other characteristics such as curvature of thearray or other aspects that could produce error of a probe array beingperfectly flat in a plane that is perpendicular to the plane of galvoarm rotation 149. In the present example, the measured calibration arraycould be placed in scanner 400 and used to calibrate the instrument tocorrect for roll and pitch error associated with the mechanicalcomponents of scanner 400.

In an alternative example, calibration features 442 could be disposeddirectly upon transport frame 505 such that they could be imaged whenprobe array cartridge 720 is not present. A durable fluorescent materialthat is resistant to wear and what is commonly referred to as photobleaching could be used as a medium for the calibration features.

In some implementations, calibration features 442 could include a knownconcentration of fluorophores disposed on a calibration slide orcalibration array that may be used as a reference medium. Calibrationfeatures 442 may be scanned by a plurality of scanners 400 for thepurpose of calibrating each of scanners 400 with respect to one another.In one embodiment the fluorophores on a calibration array have the samefluorescent properties as fluorophores used in biological experiments.For example, the same fluorophores used in biological experiments thatfor instance could include phycoerythrin, may be used as a fluorescentstandard on a calibration array. Alternatively, other types offluorophores may be preferable for use on a calibration array because ofa variety of factors that could include long term stability of themolecule, photobleaching characteristics, or other factors that may bedesirable for use in calibration procedures. In the present example, thealternative fluorophores could include kapton, or, alternatively, whatthose of ordinary skill in the related art refer to as quantum dotnanocrystals. The term “quantum dots” in this context generally refersto inorganic fluorescent nanocrystals and may be preferable in someimplementations because of their long term stability, low photobleachingqualities, as well as their fluorescent tunability. For example, arraysof uniformly distributed single quantum dots may be fabricated bymethods known to those of ordinary skill in the related art thatincludes what may be referred to as a self-assembly method known asspin-casting. The number of quantum dots disposed on the calibrationarray may be calculated so that the number of emitted photons offluorescent light per quantum dot may be calculated using the measuredfluorescence. The emitted photon per quantum dot calculation may then beused for gain calibration, instrument to instrument calibration, orother calibration methods.

Additionally in some embodiments, measured intensity error may beacquired using methods known to those of ordinary skill in the art forthe measurement of the amount of noise from the photo multiplier tube(as described above in reference to the auto-zero operation),measurement of the quantum efficiency of the laser, or measurements ofthe transmission efficiency of the dichroic mirror. Quantum efficiencycan be briefly defined as a measurement of the ratio of the energyoutput versus the energy input to the laser, where the lower the ratiovalue the more efficient the laser is. The transmission efficiency ofthe dichroic mirror similarly could be defined as a measurement of theamount of light at a particular wavelength that is allowed to passthrough or reflected by the mirror. Each component could be calibratedor replaced if need be in order to achieve the opitmal scannerperformance.

An illustrative example of one embodiment for the acquisition, storage,and method of use of linearity calibration data are provided in FIGS. 20through 24. It will be appreciated by those of ordinary skill in therelated art that the illustrative methods may be applied the both the Xaxis in the case of horizontal linearity, and the Y axis in the case ofvertical linearity. Additionally, it may be desirable in the presentembodiment to apply linearity correction methods in order to achieve astandard position error of less than +/−1 pixel. The term “positionerror” as used herein, refers to an error value associated with theplacement of pixel intensity data at the appropriate positions in animage. For example, a position error of one pixel means that theplacement of pixel intensity data should not be placed more than onepixel away from the appropriate position in an image.

An exemplary method for the creation of one or more linearity correctiontables is illustrated in FIG. 22 with an associated functional blockdiagram illustrated in FIGS. 20A, 20B, and 21. Step 2205 represents thestart point of the method where calibration image acquirer 2057initiates a line scan over calibration features 442 and samples theintensity of calibration signal 196 at regular intervals such as, forexample sampling at every 8-10 or other number of pixels. Calibrationdata generator 2050 may receive calibration signal 196 from one or morescan lines. As further illustrated in FIG. 21, pixel position determiner2110 determines the calibration feature that is closest to the centerposition of the image based, at least in part, upon galvo arm positiondata from transducer 515. Determiner 2110 may use determined centercalibration feature as a reference position.

Using similar methods as those for determining the center calibrationfeature, pixel position determiner 2110 determines the pixel positionsof the leading edge of each calibration feature relative to the centerposition, illustrated as step 2215. Step 2220 illustrates a step wherepixel position correlator 2120 correlates the determined pixel positionsfrom step 2215 with the actual position data of the corresponding edgesof each calibration feature, stored in calibration feature data 2030.Position and error data table generator 2150 calculates the differencebetween the determined and actual pixel positions as error data for theleading edge of each feature, and constructs intermediate calibrationdata 2153, as illustrated in step 2230.

As illustrated in step 2240, intermediate calibration data 2153 may bereceived by pixel offset determiner 2160. Determiner 2160 may use thedetermined center pixel position of data 2153 to assign pixel offsetpositions for each pixel position in the table. The pixel offsetpositions may be identified based upon their positional relationship inthe (+) or (−) directions from the reference point. In the presentexample the (−) direction is towards the start position of the scan, andthe (+) direction is towards the end position, although it will beappreciated by those of ordinary skill in the relevant art that otheralternatives exist.

In some embodiments, as described in the example above, the reflectedintensity may be sampled at regular intervals (i.e. every 8-10 pixels)thus data 2153 may not contain a pixel offset value and associated errorcorrection value representing each possible pixel position. Oneembodiment of calibration data table 2010 includes an error correctionvalue corresponding to each possible pixel offset position. Thedetermined pixel offset positions and associated error correction valuesin data 2153 may be received by pixel and error correction interpolator2270 as illustrated in step 2250. Interpolator 2270 may interpolateerror correction values for the pixel offset positions not contained indata 2153. For example, data 2153 may contain pixel offset positions 0,−5, −12, and −21 each having an associated error correction value. Inthe present example, the associated error correction values for pixeloffset positions −1 through −4 must be interpolated based, at least inpart, upon the associated error correction values for the pixel offsetpositions 0 and −5 of data 2153. Whereas a different scale ofinterpolated error correction values may be calculated corresponding topixel offset positions −6 through −11, based, at least in part, upon thegreater range of pixel offset positions and associated error correctionvalues. Therefore there may not be linear relationship between the errorcorrection values in calibration data 2010. Additionally, errorcorrection values may include partitioning fractions of pixel intensityvalues into different corrected pixel locations. In the present examplethe resulting data may be presented in calibration data table 2010,comprising a pixel offset position and an associated error correctionvalue for any implementation of probe array 240.

In one embodiment calibration table 2010 may be used by one or moremethods to correct image data for what is referred to as spatialnon-linearity due to error in the scanner that could come from a varietyof sources. An exemplary method is presented in FIG. 23 includingelements illustrated in FIG. 24. Scan width and zero position determiner2420 may receive table 2010 as illustrated in step 2310. In theillustrative method it may be desirable to reassign a range of the pixeloffset position values so that the 0 pixel position corresponds to thestart point of the X-axis scan. For instance, the range of pixel offsetpositions may correspond to a particular implementation of probe array240 that may have different X-axis widths. In the presently describedembodiment it is important to apply the appropriate error correctionvalue to each pixel position of each implementation of probe array 240.The width of the scan in the X-axis for a particular implementation ofprobe array 240 may be determined from scan width data 2040 based, atleast in part, upon one or more identifiers. Determiner 2420 calculatesthe value of half the total number of pixel positions of the scan widthof probe array 240 and adds that value to all of the pixel offset valuesin calibration data table 2010 starting from the center position. Theresult is that the start point of the scan that formerly had a negativevalue (i.e. was on the (−) side of the center point), is now at the zeropixel position. For example, calibration data table 2010 contains arange of pixel offset positions that is greater than the scan width ofany implementation of probe array 240. Additionally, table 2010 may havea center reference point with negative values to one side and positivevalues to the other. In the present example there may be a variety ofdifferent scan widths each associated with a particular implementationof probe array 240, where the distance between the start and end pointsof the scan vary for each particular array scan width with respect tothe center point. The start point for each scan should, in the presentexample, begin at pixel position 0. The pixel position values may becalculated by adding half the value of the scan width to the negativepixel offset values where pixel position 0 then corresponds to the startpoint of the scan width and the end point is equal to the total value ofthe scan width. The resulting pixel positions and corresponding errorcorrection values are illustrated in FIG. 24 as pixel position and errorcorrection data table 2470.

Corrected image generator 2430 receives data table 2470 for use in imagecorrection with respect to a particular implementation of probe array240. As illustrated in step 2340, a line of scan data in the X-axis isacquired from probe array 240. Generator 2430 compares each pixelposition in the acquired line of data to table 2470 and places theassociated intensity value for the acquired pixel position in acorrected pixel position in an image, as illustrated in step 2350. Insome implementations intensity values may be split among a plurality ofcorrected pixel positions. For example the error correction value for aparticular acquired pixel position may specify that the associatedintensity value may be divided between two or more corrected pixelpositions. In the present example, the intensity value may be divided bysome percentage and a portion of the intensity assigned to one correctedpixel position and the remainder is assigned to another corrected pixelposition.

Decision element 2360 illustrates the step that repeats the step 2350 ifthe image has not been completely scanned. Otherwise the scan has beencompleted, and the result is corrected image data 2070.

In some implementations it may be advantageous to use galvo arm positiondata in the place of pixel position data to calculate the errorcorrection table. Galvo arm position data may provide a greater range ofvalues that may allow for more precise image correction. In the examplesillustrated in FIGS. 20 through 24, transducer 515 may input data intocalibration data generator 2050 and pixel and corrected image generator2430. In the previously illustrated examples a point of pixel data maybe sampled at every 8-10 pixel positions. In an alternative example, theexact position of galvo arm 200 may be taken at the same or other pointsusing transducer 515. The resulting galvo positions may be used in theplace of pixel positions for the production of calibration data 2010.Similarly, galvo position data may be substituted for pixel positiondata in the offset and interpolation calculations. Corrected imagegenerator 2430 may then use the position signal from transducer 515corresponding to the position of galvo arm 200 during the acquisition ofa line of scan data to calculate the corrected pixel placements for theimage data.

Alternatively, calibration executables and data 676 may use both thegalvo position data and the pixel position data for calibration anderror correction methods. In some implementations the galvo positiondata may be used to pre-correct the image data prior to the describedimage correction procedure using the pixel position data. For example,one method could include acquiring a line of new galvo position dataprior to or during the acquisition of a line of image data. The newgalvo position data may be compared with galvo/pixel position data incalibration data 2010. Any differences between the new galvo positiondata and the data of calibration data 2010 may represent changes in themechanism since the last calibration and could be corrected by a varietyof methods.

Another embodiment of a method using calibration features 442 mayinclude calculating the radius of the arc of arcuate path 250. In manyimplementations it may be desirable to have the exact radius distance ofthe path of excitation beam 135 from the center of the axis of rotationof galvo arm 200 that may, for instance, be used to calculate an errorvalue based, at least in part upon the difference between the actualradius and the expected or optimal radius that could include a radius of25.4 mm. The radius value may be particularly useful for some imagecorrection methods including arc correction methods that will bedescribed in greater detail below. An illustrated example is presentedin FIG. 25 of scanning arm 200 and two possible paths of excitation beam135. Expected light path 2510 represents the expected path of excitationbeam 135 emitted from center of lens 145 that meets the substrate at a90° angle resulting in the expected radius R 3100. Radius R′ 2505represents a graphical illustration of an example where the actualradius is different than the expected radius due to actual light path2515. Actual light path 2515 could be due to misalignment orimperfections in turning mirrors, lens 145, or a variety of otherfactors that may affect the optical path.

An exemplary method of determining radius R′ 2505 is illustrated in FIG.27 with a graphical depiction of particular elements illustrated inFIGS. 20 and 26. The method begins with the acquisition of an image ofcalibration features 442 as illustrated in step 2710 that image beingreceived by radius distance generator 2055. Calibration features 442 mayinclude a variety of possible implementations such as, for instance, thepreviously described ladder array of chrome features in reference tohorizontal linearity. Generator 2055 determines the pixel position thatcorresponds to the leading edge of a calibration feature located in thecenter of the arc of arcuate path 250 and assigns that position as datapoint 2615. Step 2720 of the method includes generator 2055 moves adistance 2605 in both directions along the X-axis away from the centerpoint 2615. Distance 2605 can be any distance within the scan arc thatcorresponds to the leading edge of a calibration feature, but one methodmay include distance 2605 that is near the edges of the scan arc. Datapoints 2610 and 2617 are established at the positions corresponding tocalibration features distance 2605 away in both directions on theX-axis. Generator 2055 may normalize the positions of data points 2610and 2617 with respect to the Y-axis so that they are in the same planeas illustrated as step 2725. The normalized positions of data points2610 and 2617 may, in some implementations, be used to correct for anyrotation of the calibration features present in the acquired image. Thenormalization process may include a variety of mathematical methodsincluding the determination of the slope of line 2640 drawn betweenpoints 2610 and 2617. Those of ordinary skill in the related art willappreciate that a slope value is zero indicating that points 2610 and2617 are in the same plane on the Y-axis. Generator 2055 may move one ormore of points 2610 and 2617, while maintaining distance 2605, until theslope value is obtained. Step 2730 illustrates the step where generator2055 mathematically calculates arc 2642 for the best fit to data points2610, 2617, and 2615. The actual radius R′ 2505 is then determined fromthe center point of rotation 2520 to arc 2642. The calculated actualradius 2505 may be stored in calibration feature data 2030 or otherlocations for use in various error correction methods, such as themethod of radius adjustment described in greater detail below withrespect to tool 3000.

Some aspects of scanner calibration and configuration could be carriedout by service application 678 that may be implemented by scannerfirmware executables 672 or alternatively by scanner control andanalysis executables 572. In the example illustrated in FIG. 6 in isshown as being located in system memory 670 of scanner computer 510 butalternatively may be located in computer 350 or as a stand aloneapplication included in a local or remote computer connected via thenetwork connection of input-output controllers 675 or other means ofconnection. Service personnel could use service application 678 at thefactory for initial calibration and/or in the field during routine orother types of service. In one embodiment, the service application mayact as a low level interface to all of the hardware components ofscanner 400. For example, service application 678 could perform all ofthe functions required for calibration that could include but are notlimited to setting the numbers and frequencies of the lasers, laserpower settings, emission detector settings, as well as the previouslydescribed methods. In the present example, service application 678 couldalso run diagnostic tests and upload/download software and firmware asneeded to scanner computer 510.

Additional elements of service application 678 could include elementsthat enable a user to perform installation and initial calibrationprocedures of scanner 400. For example, the elements could allow a userto receive a scanner from a remote source and perform the necessary setup operations in a reasonable period of time, such as one hour or less.In the present example, the scanner is completely calibrated andaligned, and fully capable of normal operation after the userinstallation.

Some embodiments of the invention may also include hardware elementsused for calibration. One such element is referred to herein as tool3000, illustrative examples of which are illustrated in FIGS. 30A and30B. Tool 3000 may be used for initial instrument calibration at thefactory or in the field by instrument technicians or users. Tool 3000enables the precise calibration, measurement, and adjustment of avariety of elements of scanner 400, including the Y 707 axis distancethat for instance may correspond to the expected radius R 2500 of FIG.25, and the rotational alignment of galvo arm 200 that may include theperpendicular alignment of excitation beam 135 to probe array 240. Tool3000 may, in some embodiments, clamp onto and/or be firmly seated upongalvo arm 200. For example, tool 3000 may include galvo arm cutout 3007that may have a shape corresponding to the outside shape of galvo arm200. Cap 3005 may include a first half of cutout 3007 and handle 3010may include the second half. In the present example, galvo arm 200 maybe firmly held by tool 3000 by the placement of handle 3010 on thesecond half side of galvo arm 200 and attaching cap 3005 to the firsthalf side of handle 3010 over galvo arm 200. In the present examplegalvo arm 200 may have one or more fiducial features that only allowattachment of tool 3000 to galvo arm 200 in a manner so thatmeasurements of rotation and Y-axis distance may be reliably andrepeatedly measured.

Fixed mounting plate 3015 may provide one or more reference positionsused for calibration adjustments or settings. In one embodiment, fixedmounting plate 3015 may be a fixed rigid structure within scanner 400capable of interacting with tool 3000. Alternatively, plate 3015 may beattached to scanner 400 via commonly used methods each time tool 3000may be used for calibration or measurement methods. Tool 3000 may use aplurality of measuring devices for implementing methods for calibrationand measurement that could include rotational micrometer 3030 that maybe held in place by micrometer bracket 3020, and Y-axis micrometer 3035that may be held in place by cap 3005. Both micrometers 3030 and 3035may interact directly with elements of scanner 400 and/or plate 3015 forthe purposes of precision calibration and/or measurement. For example,calibration executables and data 676 may execute a line scan overcalibration features 442 and determine one or more error valuescorresponding to the rotational axis and Y-axis distance of galvo arm200. Alternatively, calibration executables and data 676 may be enabledto acquire the error values from a line scan over probe array 240. Tool3000 may be attached to galvo arm 200 so that handle 3010 and cap 3005have rotational and Y-axis freedom of movement in relation to fixedmounting plate 3015. A user may initially use micrometers 3030 and 3035to precisely measure the rotational and Y-axis values. The user may thenrelease galvo arm 200 such as, for instance, by releasing a clamp orother means of holding, and use micrometers 3030 and 3035 to apply theone or more error correction values determined by calibrationexecutables and data 676. In the present example micrometers 3030 and3035 may directly apply the methods of error correction by pushinghandle 3010 and/or cap 3005 in a first direction that may includeincreasing the distance from micrometers 3030 and/or 3035, oralternatively allowing the spring rate of one or more elements to bringhandle 3010 and/or cap 3005 closer (i.e. decreasing the distance tomicrometers 3030 and 3035). The user may reapply the means of holdinggalvo arm 200 and verify the values on micrometers 3030 and 3035 andrelease tool 3000.

Having described various embodiments and implementations, it should beapparent to those skilled in the relevant art that the foregoing isillustrative only and not limiting, having been presented by way ofexample only. Many other schemes for distributing functions among thevarious functional elements of the illustrated embodiment are possible.The functions of any element may be carried out in various ways inalternative embodiments.

Also, the functions of several elements may, in alternative embodiments,be carried out by fewer, or a single, element. Similarly, in someembodiments, any functional element may perform fewer, or different,operations than those described with respect to the illustratedembodiment. Also, functional elements shown as distinct for purposes ofillustration may be incorporated within other functional elements in aparticular implementation. Also, the sequencing of functions or portionsof functions generally may be altered. Certain functional elements,files, data structures, and so on may be described in the illustratedembodiments as located in system memory of a particular computer. Inother embodiments, however, they may be located on, or distributedacross, computer systems or other platforms that are co-located and/orremote from each other. For example, any one or more of data files ordata structures described as co-located on and “local” to a server orother computer may be located in a computer system or systems remotefrom the server. In addition, it will be understood by those skilled inthe relevant art that control and data flows between and amongfunctional elements and various data structures may vary in many waysfrom the control and data flows described above or in documentsincorporated by reference herein. More particularly, intermediaryfunctional elements may direct control or data flows, and the functionsof various elements may be combined, divided, or otherwise rearranged toallow parallel processing or for other reasons. Also, intermediate datastructures or files may be used and various described data structures orfiles may be combined or otherwise arranged. Numerous other embodiments,and modifications thereof, are contemplated as falling within the scopeof the present invention as defined by appended claims and equivalentsthereto.

1. A scanning system, comprising: one or more optical elementsconstructed and arranged to direct a excitation beam at a probe array;one or more detectors constructed and arranged to receive reflectedintensity data responsive to the excitation beam, wherein the reflectedintensity data is responsive, at least in part, to a focusing distancebetween an optical element and the probe array; a transport frameconstructed and arranged to adjust the focusing distance in a firstdirection with respect to the probe array; an auto-focuser constructedand arranged to determine a best plane of focus based, at least in part,upon one or more characteristics of the reflected intensity data asreceived at two or more focusing distances; and wherein: the one or moredetectors are further constructed and arranged to receive a plurality ofpixel intensity values based, at least in part, upon detected emissionsfrom a plurality of probe features disposed on the probe array at thebest plane of focus; and the system further comprises an image generatorconstructed and arranged to associate each of the pixel intensity valueswith one or more image pixel positions of a probe array based, at leastin part, upon one or more position correction values. 2-107. (canceled)